Galleria mellonellacationic protein 8 (GmCP8) is a hemolymph protein previously identified as an opsonin and an inhibitor of fungal proteases. In this work, we showed its bactericidal activity towardPseudomonas entomophila,Pseudomonas aeruginosa,Bacillus thuringiensis,Staphylococcus aureus, andEscherichia coliand against yeast-like fungiCandida albicans. The activity againstE. coliwas correlated with bacterial membrane permeabilization. In turn, in the case ofP. entomophila,B. thuringiensis,andC. albicans, the atomic force microscopy analysis of the microbial surface showed changes in the topography of cells and changes in their nanomechanical properties. GmCP8 also showed the inhibitory activity toward the serine protease trypsin and the metalloproteinase thermolysin. The expression of the gene encoding the GmCP8 protein did not increase either in the gut or in the fat body ofG. mellonellaafter oral infection withP. entomophila. Similarly, the amount of GmCP8 in the hemolymph ofG. mellonelladid not change in immune-challenged insects. However, when GmCP8 was injected into theG. mellonellahemocel, a change in the survival curve was observed in the infected larvae. Our results shed new light on the function of GmCP8 protein in insect immunity, indicating its role in humoral defence mechanisms.

Purpose: The zoonotic opportunistic pathogen Staphylococcus pseudintermedius 222 produces BacSp222 – an atypical peptide exhibiting the features of a bacteriocin, a virulence factor, and a molecule modulating the host inflammatory reaction. The peptide is secreted in an unmodified form and, additionally, two forms modified posttranslationally by succinylation. This study is a comprehensive report focusing on the proinflammatory properties of such molecules. Methods: The study was performed on mouse monocyte/macrophage-like and endothelial cell lines as well as human neutrophils. The following peptides were studied: BacSp222, its succinylated forms, the form deprived of formylated methionine, and a reference bacteriocin – nisin. The measurements of the nitric oxide (NO) level, induced NO synthase (iNOS) expression, the profile of secreted cytokines, NF-kappa-B activation, reactive oxygen species (ROS) biosynthesis, and the formation of extracellular traps were conducted to evaluate the proinflammatory activity of the studied peptides. Results: BacSp222 and its succinylated forms effectively induced NO production and iNOS expression when combined with IFN-gamma in macrophage-like cells. All natural BacSp222 forms used alone or with IFN-gamma stimulated the production of TNF-alpha, MCP-1, and IL-1-alpha, while the co-stimulation with IFN-gamma increased IL-10 and IL-27. Upregulated TNF-alpha secretion observed after BacSp222 exposition resulted from increased expression but not from membrane TNF-alpha proteolysis. In neutrophils, all forms of bacteriocin upregulated IL-8, but did not induce ROS production or NETs formation. In all experiments, the activities of deformylated bacteriocin were lower or unequivocal in comparison to other forms of the peptide. Conclusion: All naturally secreted forms of BacSp222 exhibit proinflammatory activity against monocyte-macrophage cells and neutrophils, confirming that the biological role of BacSp222 goes beyond bactericidal and cytotoxic effects. The atypical posttranslational modification (succinylation) does not diminish its immunomodulatory activity in contrast to the lower antibacterial potential or cytotoxicity of such modified form established in previous studies.

Red palm weevil (Rhynchophorus ferrugineus Olivier, 1791, Coleoptera: Curculionidae) is a destructive pest of palms, rapidly extending its native geographical range and causing large economic losses worldwide. The present work describes isolation, identification, and bioinformatic analysis of antibacterial proteins and peptides from the immunized hemolymph of this beetle. In total, 17 different bactericidal or bacteriostatic compounds were isolated via a series of high-pressure liquid chromatography steps, and their partial amino acid sequences were determined by N-terminal sequencing or by mass spectrometry. The bioinformatic analysis of the results facilitated identification and description of corresponding nucleotide coding sequences for each peptide and protein, based on the recently published R. ferrugineus transcriptome database. The identified compounds are represented by several well-known bactericidal factors: two peptides similar to defensins, one cecropin-A1-like peptide, and one attacin-B-like protein. Interestingly, we have also identified some unexpected compounds comprising five isoforms of pheromone-binding proteins as well as seven isoforms of odorant-binding proteins. The particular role of these factors in insect response to bacterial infection needs further investigation.

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.

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.

Collagen-hyaluronic acid (Col-HA) hydrogels are widely studied as biomimetic materials that recapitulate the environmental physical and mechanical properties crucial for understanding the cell behavior during cancer invasion and progression. Our research focused on Col-HA hydrogels as an environment to study the invasion of bladder cancer cells through the bladder wall. The bladder is a heterogeneous structure composed of three main layers: urothelium (the softest), lamina propria (the stiffest), and the muscle outer layer, with elastic properties lying between the two. Thus, the bladder cancer cells migrate through the mechanically distinct environments. We investigated the impact of Col-HA hydrogel microstructure and rheology on migrating bladder cancer T24 cells from the cancer spheroid surface to the surrounding environment formed from various collagen I and HA concentrations and chemical structures. The designed hydrogels showed variability in network density and rheological properties. The migration of bladder cancer cells was inhibited inside hydrogels of ∼1 kPa storage modulus. The correlation analysis showed that collagen concentration primarily defined the rheological properties of Col-HA hydrogels, but hydrogels can soften or stiffen depending on the type of HA used. Within soft Col-HA hydrogels, cells freely invade the surrounding environment, while its stiffening impedes cell movement and almost inhibits cell migration. Only individual, probably leading, cells are observed at the spheroid edges initiating the invasion. Our findings showed that the rheological properties of the hydrogels dominate in regulating cancer cell migration, providing a platform to study how bladder cancer cells migrate through the heterogeneous structure of the bladder wall.