Despite the odds: formation of the SARS-CoV-2 methylation complex.

Coronaviruses modify their single-stranded RNA genome with a methylated cap during replication to mimic the eukaryotic mRNAs. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16, while nsp10 acts as a co-factor to both. Additionally, nsp14 carries an exonuclease domain which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins would be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex upon significant allosteric change. This interaction is expected to encourage the formation of mature capped viral mRNA, modulating nsp14's exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.

BacSp222 bacteriocin as a novel ligand for TLR2/TLR6 heterodimer.

OBJECTIVE AND DESIGN: BacSp222 bacteriocin is a bactericidal and proinflammatory peptide stimulating immune cells to produce selected cytokines and NO in NF-ĸB dependent manner. This study aims to identify the receptor which mediates this activity. METHODS: We applied fluorescently labeled BacSp222 and a confocal microscopy imaging to analyze the direct interaction of the bacteriocin with the cells. Reporter HEK-Blue cells overexpressing human toll-like receptors (TLR2, TLR4, TLR5 or TLR2/TLR1 and TLR2/TLR6 heterodimers) were stimulated with BacSp222, and then the activity of NF-ĸB-dependent secreted embryonic alkaline phosphatase (SEAP) was measured. In turn, formylated peptide receptor (FPR) or TLR2 antagonists were used to verify bacteriocin-stimulated TNF production by murine monocyte-macrophage cell lines. RESULTS: BacSp222 undergoes internalization into cells without disturbing the cell membrane. FPR antagonists do not affect TNF produced by BacSp222-stimulated murine macrophage-like cells. In contrast, BacSp222 stimulates NF-ĸB activation in HEK-Blue overexpressing TLR2 or TLR2/TLR6 heterodimer, but not TLR2/TLR1, TLR4 or TLR5 receptors. Moreover, TLR2-specific antagonists inhibit NF-ĸB signaling in BacSp222-stimulated HEK-Blue TLR2/TLR6 cells and reduce TNF release by BacSp222-treated RAW 264.7 and P388.D1. CONCLUSIONS: BacSp222 is a novel ligand for TLR2/TLR6 heterodimer. By binding TLR complex the bacteriocin undergoes internalization, inducing proinflammatory signaling that employs MyD88 and NF-ĸB pathways.

Structure, Biosynthesis, and Biological Activity of Succinylated Forms of Bacteriocin BacSp222.

BacSp222 is a multifunctional peptide produced by Staphylococcus pseudintermedius 222. This 50-amino acid long peptide belongs to subclass IId of bacteriocins and forms a four-helix bundle molecule. In addition to bactericidal functions, BacSp222 possesses also features of a virulence factor, manifested in immunomodulatory and cytotoxic activities toward eukaryotic cells. In the present study, we demonstrate that BacSp222 is produced in several post-translationally modified forms, succinylated at the ε-amino group of lysine residues. Such modifications have not been previously described for any bacteriocins. NMR and circular dichroism spectroscopy studies have shown that the modifications do not alter the spatial structure of the peptide. At the same time, succinylation significantly diminishes its bactericidal and cytotoxic potential. We demonstrate that the modification of the bacteriocin is an effect of non-enzymatic reaction with a highly reactive intracellular metabolite, i.e., succinyl-coenzyme A. The production of succinylated forms of the bacteriocin depends on environmental factors and on the access of bacteria to nutrients. Our study indicates that the production of succinylated forms of bacteriocin occurs in response to the changing environment, protects producer cells against the autotoxicity of the excreted peptide, and limits the pathogenicity of the strain.

Fungal α-1,3-Glucan as a New Pathogen-Associated Molecular Pattern in the Insect Model Host Galleria mellonella.

Recognition of pathogen-associated molecular patterns (PAMPs) by appropriate pattern recognition receptors (PRRs) is a key step in activating the host immune response. The role of a fungal PAMP is attributed to ß-1,3-glucan. The role of α-1,3-glucan, another fungal cell wall polysaccharide, in modulating the host immune response is not clear. This work investigates the potential of α-1,3-glucan as a fungal PAMP by analyzing the humoral immune response of the greater wax moth Galleria mellonella to Aspergillus niger α-1,3-glucan. We demonstrated that 57-kDa and 61-kDa hemolymph proteins, identified as ß-1,3-glucan recognition proteins, bound to A. niger α-1,3-glucan. Other hemolymph proteins, i.e., apolipophorin I, apolipophorin II, prophenoloxidase, phenoloxidase activating factor, arylphorin, and serine protease, were also identified among α-1,3-glucan-interacting proteins. In response to α-1,3-glucan, a 4.5-fold and 3-fold increase in the gene expression of antifungal peptides galiomicin and gallerimycin was demonstrated, respectively. The significant increase in the level of five defense peptides, including galiomicin, corresponded well with the highest antifungal activity in hemolymph. Our results indicate that A. niger α-1,3-glucan is recognized by the insect immune system, and immune response is triggered by this cell wall component. Thus, the role of a fungal PAMP for α-1,3-glucan can be postulated.

Choline Supplementation Sensitizes Legionella dumoffii to Galleria mellonella Apolipophorin III.

The growth of Legionella dumoffii can be inhibited by Galleria mellonella apolipophorin III (apoLp-III) which is an insect homologue of human apolipoprotein E., and choline-cultured L. dumoffii cells are considerably more susceptible to apoLp-III than bacteria grown without choline supplementation. In the present study, the interactions of apoLp-III with intact L. dumoffii cells cultured without and with exogenous choline were analyzed to explain the basis of this difference. Fluorescently labeled apoLp-III (FITC-apoLp-III) bound more efficiently to choline-grown L. dumoffii, as revealed by laser scanning confocal microscopy. The cell envelope of these bacteria was penetrated more deeply by FITC-apoLp-III, as demonstrated by fluorescence lifetime imaging microscopy analyses. The increased susceptibility of the choline-cultured L. dumoffii to apoLp-III was also accompanied by alterations in the cell surface topography and nanomechanical properties. A detailed analysis of the interaction of apoLp-III with components of the L. dumoffii cells was carried out using both purified lipopolysaccharide (LPS) and liposomes composed of L. dumoffii phospholipids and LPS. A single micelle of L. dumoffii LPS was formed from 12 to 29 monomeric LPS molecules and one L. dumoffii LPS micelle bound two molecules of apoLp-III. ApoLp-III exhibited the strongest interactions with liposomes with incorporated LPS formed of phospholipids isolated from bacteria cultured on exogenous choline. These results indicated that the differences in the phospholipid content in the cell membrane, especially PC, and LPS affected the interactions of apoLp-III with bacterial cells and suggested that these differences contributed to the increased susceptibility of the choline-cultured L. dumoffii to G. mellonella apoLp-III.

Antifungal Activity of Anionic Defense Peptides: Insight into the Action of Galleria mellonella Anionic Peptide 2.

Anionic antimicrobial peptides constitute an integral component of animal innate immunity, however the mechanisms of their antifungal activity are still poorly understood. The action of a unique Galleria mellonella anionic peptide 2 (AP2) against fungal pathogen Candida albicans was examined using different microscopic techniques and Fourier transform infrared (FTIR) spectroscopy. Although the exposure to AP2 decreased the survival rate of C. albicans cells, the viability of protoplasts was not affected, suggesting an important role of the fungal cell wall in the peptide action. Atomic force microscopy showed that the AP2-treated cells became decorated with numerous small clods and exhibited increased adhesion forces. Intensified lomasome formation, vacuolization, and partial distortion of the cell wall was also observed. FTIR spectroscopy suggested AP2 interactions with the cell surface proteins, leading to destabilization of protein secondary structures. Regardless of the anionic character of the whole AP2 molecule, bioinformatics analyses revealed the presence of amphipathic α-helices with exposed positively charged lysine residues. High content of the α-helical structure was confirmed after deconvolution of the IR absorption spectrum and during circular dichroism measurements. Our results indicated that the antimicrobial properties of G. mellonella AP2 rely on the same general characteristics found in cationic defense peptides.

Studies on the interactions of neutral Galleria mellonella cecropin D with living bacterial cells.

Cecropins constitute an important family of insect antimicrobial peptides involved in humoral innate immune response. In comparison with the highly basic cecropins A and B, cecropins D are less cationic and more hydrophobic. Interestingly, cecropins D were described only in lepidopteran insects, e.g., the greater wax moth Galleria mellonella. In the present study, interactions of neutral cecropin D (pI 6.47) purified from hemolymph of G. mellonella with living Escherichia coli cells were investigated. Fluorescence lifetime imaging microscopy using fluorescein isothiocyanate-labeled cecropin D revealed very fast binding of the peptide to E. coli cells. Fourier transform infrared spectroscopy analyses showed that G. mellonella cecropin D interacted especially with E. coli LPS and probably other lipid components of the bacterial cell envelope and exhibited an ordering effect with regard to lipid chains. This effect is consistent with the peptide binding mechanism based upon its incorporation into the lipid phase of the cell membrane. The interaction resulted in permeabilization of the bacterial cell membrane. Upon cecropin D binding, the cells lost characteristic surface topography, which was accompanied by altered nanomechanical properties, as revealed by atomic force microscopy. The interaction of the peptide with the bacterial cells also led to intracellular damage, i.e., loss of the cell envelope multilayer structure, formation of membrane vesicles, and enlargement of periplasmic space, which eventually caused death of the bacteria. In summary, it can be concluded that amphipathic character of α-helices, exposure of small positively charged patches on their polar surfaces and hydrophobic interactions are important physicochemical characteristics related to effective binding to E. coli cells and antibacterial activity of neutral G. mellonella cecropin D.

Identification of Antioxidant Peptides in Enzymatic Hydrolysates of Carp (Cyprinus Carpio) Skin Gelatin.

The protein by-products from carp (Cyprinus carpio) are normally discarded as industrial waste during fish processing. The objective of this study was to identify and characterise the peptides with a potential antioxidant activity that are released from carp skin proteins during hydrolysis by the Protamex enzyme mixture. This study shows that a hydrolysate of carp skin gelatin and its reversed-phase chromatography fractions have strong in vitro antioxidant properties. Among these fractions, the alanine-tyrosine (Ala-Tyr) dipeptide was identified as the major compound with high antioxidant potential. The peptide has good stability during in vitro enzymatic digestion assay and can inhibit the angiotensin-converting enzyme (ACE). In conclusion, our study proves that both the unfractionated hydrolysate of carp skin gelatin and the above-mentioned Ala-Tyr dipeptide represents attractive novel compounds for the formulation of antioxidant foods.

The lipid composition of Legionella dumoffii membrane modulates the interaction with Galleria mellonella apolipophorin III.

Apolipophorin III (apoLp-III), an insect homologue of human apolipoprotein E (apoE), is a widely used model protein in studies on protein-lipid interactions, and anti-Legionella activity of Galleria mellonella apoLp-III has been documented. Interestingly, exogenous choline-cultured Legionella dumoffii cells are considerably more susceptible to apoLp-III than non-supplemented bacteria. In order to explain these differences, we performed, for the first time, a detailed analysis of L. dumoffii lipids and a comparative lipidomic analysis of membranes of bacteria grown without and in the presence of exogenous choline. (31)P NMR analysis of L. dumoffii phospholipids (PLs) revealed a considerable increase in the phosphatidylcholine (PC) content in bacteria cultured on choline medium and a decrease in the phosphatidylethanolamine (PE) content in approximately the same range. The interactions of G. mellonella apoLp-III with lipid bilayer membranes prepared from PLs extracted from non- and choline-supplemented L. dumoffii cells were examined in detail by means of attenuated total reflection- and linear dichroism-Fourier transform infrared spectroscopy. Furthermore, the kinetics of apoLp-III binding to liposomes formed from L. dumoffii PLs was analysed by fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy using fluorescently labelled G. mellonella apoLp-III. Our results indicated enhanced binding of apoLp-III to and deeper penetration into lipid membranes formed from PLs extracted from the choline-supplemented bacteria, i.e. characterized by an increased PC/PE ratio. This could explain, at least in part, the higher susceptibility of choline-cultured L. dumoffii to G. mellonella apoLp-III.

Highly amyloidogenic two-chain peptide fragments are released upon partial digestion of insulin with pepsin.

Proteases play a well recognized role in the emergence of highly aggregation-prone protein fragments in vivo, whereas in vitro limited proteolysis is often employed to probe different phases of amyloidogenic pathways. Here, we show that addition of moderate amounts of pepsin to acidified bovine insulin at close to physiological temperature results in an abrupt self-assembly of amyloid-like fibrils from partially digested insulin fragments. Biochemical analysis of the pepsin-induced fibrils implicates peptide fragments (named H) consisting of the 13 or 15 N-terminal residues of the A-chain and 11 or 13 N-terminal residues of the B-chain linked by the disulfide bond between Cys-7A-Cys-7B as the main constituents. There are up to eight pepsin-cleavage sites remaining within the double chain peptide, which become protected upon fast fibrillation unless concentration of the enzyme is increased resulting in complete digestion of insulin. Controlled re-association of H-peptides leads to "explosive" fibrillation only under nonreducing conditions implying the key role of the disulfide bond in their amyloidogenicity. Such re-assembled amyloid is similar in terms of morphology and infrared features to typical bovine insulin fibrils, although it lacks the ability to seed the intact protein.

Physicochemical Studies on Orientation and Conformation of a New Bacteriocin BacSp222 in a Planar Phospholipid Bilayer.

The behavior, secondary structure, and orientation of a recently discovered bacteriocin-like peptide BacSp222 in a lipid model system supported at a gold electrode was investigated by chronocoulometry, polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS), and attenuated total reflectance infrared (ATR-IR) spectroscopy. The IR spectra show that the secondary structure of BacSp222 is predominantly α-helical. Analysis of the spectra in the amide I region shows that the α-helical fragment of the peptide is inserted into bilayer at the potential range at which the bilayer is stable and attached to the Au(111) surface, i.e., from -0.5 to 0.3 V vs Ag/AgCl. Insertion of BacSp222 to the membrane significantly changes the conformation of the acyl chains of lipid molecules, from all-trans to partially melted; however, the chains become less tilted. Based on these results, we propose that BacSp222 interacts with the DMPC bilayer through the barrel-stave pore formation. In this model, α-helix of BacSp222 inserts into the membrane with an angle between the α-helix axis and membrane normal equal to ∼18°. The changes in orientation of the α-helical fragment of the peptide indicate that the orientation of BacSp222 with respect to the bilayer surface is potential-dependent. The peptide is inserted into the membrane driven by the electrostatic field generated by negative charge at the metal surface. It is not inserted at negative potentials where the membrane is detached from the metal and no longer exposed to the electrostatic field of the metal.

Staphylococcal SplB serine protease utilizes a novel molecular mechanism of activation.

Staphylococcal SplB protease belongs to the chymotrypsin family. Chymotrypsin zymogen is activated by proteolytic processing at the N terminus, resulting in significant structural rearrangement at the active site. Here, we demonstrate that the molecular mechanism of SplB protease activation differs significantly and we characterize the novel mechanism in detail. Using peptide and protein substrates we show that the native signal peptide, or any N-terminal extension, has an inhibitory effect on SplB. Only precise N-terminal processing releases the full proteolytic activity of the wild type analogously to chymotrypsin. However, comparison of the crystal structures of mature SplB and a zymogen mimic show no rearrangement at the active site whatsoever. Instead, only the formation of a unique hydrogen bond network, distant form the active site, by the new N-terminal glutamic acid of mature SplB is observed. The importance of this network and influence of particular hydrogen bond interactions at the N terminus on the catalytic process is demonstrated by evaluating the kinetics of a series of mutants. The results allow us to propose a consistent model where changes in the overall protein dynamics rather than structural rearrangement of the active site are involved in the activation process.

Galleria mellonella apolipophorin III - an apolipoprotein with anti-Legionella pneumophila activity.

The greater wax moth Galleria mellonella has been exploited worldwide as an alternative model host for studying pathogenicity and virulence factors of different pathogens, including Legionella pneumophila, a causative agent of a severe form of pneumonia called Legionnaires' disease. An important role in the insect immune response against invading pathogens is played by apolipophorin III (apoLp-III), a lipid- and pathogen associated molecular pattern-binding protein able to inhibit growth of some Gram-negative bacteria, including Legionella dumoffii. In the present study, anti-L. pneumophila activity of G. mellonella apoLp-III and the effects of the interaction of this protein with L. pneumophila cells are demonstrated. Alterations in the bacteria cell surface occurring upon apoLp-III treatment, revealed by Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy, are also documented. ApoLp-III interactions with purified L. pneumophila LPS, an essential virulence factor of the bacteria, were analysed using electrophoresis and immunoblotting with anti-apoLp-III antibodies. Moreover, FTIR spectroscopy was used to gain detailed information on the type of conformational changes in L. pneumophila LPS and G. mellonella apoLp-III induced by their mutual interactions. The results indicate that apoLp-III binding to components of bacterial cell envelope, including LPS, may be responsible for anti-L. pneumophila activity of G. mellonella apoLp-III.

Isolation and Characterization of Acetylated Derivative of Recombinant Insulin Lispro Produced in Escherichia coli.

PURPOSE: Insulin lispro is a rapid-acting insulin analogue produced by recombinant DNA technology. As a biosynthetic drug, the protein undergoes strict monitoring aiming for detection and characterization of impurities. The goal of this study was to isolate and identify a derivative of insulin lispro formed during biosynthesis. METHODS: For this purpose, ion exchange chromatography in combination with endoproteinase Glu-C digestion, MALDI-TOF/TOF mass spectrometry and Edman sequencing were employed. RESULTS: Ion exchange chromatography analysis of related proteins in development batches of recombinant insulin lispro revealed the existence of unknown derivative in excess of the assumed limit. Its molecular mass was 42 Da higher than the theoretical mass of Lys(B31) insulin lispro--one of the expected process-related intermediates. Endoproteinase Glu-C cleavage enabled indication of the modified peptide. Tandem mass spectrometry (MS/MS) allowed to explore the location and type of the modification. The 42 amu shift was present in the mass of y-type ions, while b-type ions were in agreement with theoretical values. It suggested that the modification is present on B31 lysine. Further inquiry revealed the presence of two diagnostic ions for lysine acetylation at m/z 143.1 and 126.1. In addition, the peptide was isolated and sequenced by Edman degradation. Standards of phenylthiohydantoin derivatives of N-ε-acetyl-L-lysine and N-ε-trimethyl-L-lysine, not available commercially, were synthesized in the laboratory. The retention time of the modified residue confirmed its identity as N-ε-acetyl-L-lysine. CONCLUSIONS: The derivative of insulin lispro formed during biosynthesis of the drug was identified to be N-ε-acetyl-L-lysine (B31) insulin lispro.

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 Young's 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.

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.

Pro-inflammatory properties of aureocin A53.

Aureocin A53 is a peptide bacteriocin produced by an opportunistic pathogen Staphylococcus aureus strain A53. The spatial structure of aureocin, unlike its amino acid sequence, is similar to the bacteriocin BacSp222, which was recently found to have the ability to induce the inflammatory response in the host cells. The presented research aimed to verify such properties also for aureocin A53. We demonstrated that the synthetic aureocin has slight cytotoxic activity towards murine monocytic-macrophage cells. This molecule was also able to activate murine P388.D1 and RAW 264.7 cells to IFN-γ-dependent production of nitric oxide and to activate production of the pro-inflammatory cytokine - TNF. We also proved that the observed pro-inflammatory activity of the studied bacteriocin is related to the stimulation of the TLR2/TLR6 heterodimer and, consequently, activation of the NF-κB transcription factor. To sum up, A53 is the second bacteriocin described in the literature, showing the pro-inflammatory activity against murine macrophage-like cells.

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.

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.

Vortex-induced amyloid superstructures of insulin and its component A and B chains.

Insulin is an amyloid-forming polypeptide built of two disulfide-linked chains (A and B), both themselves amyloidogenic. An interesting property of insulin is that agitation strongly influences the course of its aggregation, resulting in characteristic chiral superstructures of amyloid fibrils. Here, we investigate the self-assembly of these superstructures by comparing the quiescent and vortex-assisted aggregation of insulin and its individual A and B chains in the presence or absence of reducing agent tris(2-carboxyethyl)phosphine (TCEP). Our study shows that only the B chain in the presence of TCEP is converted into aggregates with morphology (according to atomic force microscopy) and optical activity (manifested as an extrinsic Cotton effect induced in bound thioflavin T) characteristic of amyloid superstructures that are normally formed by insulin in the absence of TCEP. In contrast to more rigid B-peptide fibrils, elongated aggregates of the A peptide become amorphous upon agitation. Moreover, the aggregation of equimolar mixture of both peptides does not produce highly ordered entities. Our results suggest that the dynamics of the B chain are the driving force for the assembly of superstructures, with the A chain being complicit as long as its own dynamics are controlled by the firm attachment to the B chain provided by the intact covalent structure of insulin.