Anti-Legionella dumoffii Activity of Galleria mellonella Defensin and Apolipophorin III

Abstract

The gram-negative bacterium Legionella dumoffii is, beside Legionella pneumophila, an etiological agent of Legionnaires’ disease, an atypical form of pneumonia. The aim of this study was to determine the antimicrobial activity of Galleria mellonella defense polypeptides against L. dumoffii. The extract of immune hemolymph, containing a mixture of defense peptides and proteins, exhibited a dose-dependent bactericidal effect on L. dumoffii. The bacterium appeared sensitive to a main component of the hemolymph extract, apolipophorin III, as well as to a defense peptide, Galleria defensin, used at the concentrations 0.4 mg/mL and 40 μg/mL, respectively. L. dumoffii cells cultured in the presence of choline were more susceptible to both defense factors analyzed. A transmission electron microscopy study of bacterial cells demonstrated that Galleria defensin and apolipophorin III induced irreversible cell wall damage and strong intracellular alterations, i.e., increased vacuolization, cytoplasm condensation and the appearance of electron-white spaces in electron micrographs. Our findings suggest that insects, such as G. mellonella, with their great diversity of antimicrobial factors, can serve as a rich source of compounds for the testing of Legionella susceptibility to defense-related peptides and proteins.

Cationic Antimicrobial Polymers and Their Assemblies

“…Microbial resistance mechanisms against the host defense peptides include several strategies such as inactivation and cleavage of host defense peptides by production of host defense binding proteins and proteases, repulsion of the peptides by alteration of pathogen’s surface charge employing modifications by amino acids or amino sugars of anionic molecules (e.g., teichoic acids, lipid A and phospholipids), alteration of bacterial membrane fluidity, and elimination of the peptides using multi drug efflux pumps. Together with bacterial regulatory network(s) that regulate expression and activity of these mechanisms, they represent attractive targets for development of novel antibacterials [178–180]. …”

LYSOZYME AND DEFENSE PEPTIDES AS SUPPRESSORS OF PHENOLOXIDASE ACTIVITY IN Galleria mellonella

“…The defense peptides and lysozyme were purified from the immune hemolymph extracts using a modified procedure described in our previous papers (Cytryńska et al, 2007; Archives of Insect Biochemistry and Physiology Palusinska-Szysz et al, 2012;Zdybicka-Barabas et al, 2012) using a P680 HPLC system (Dionex, Munchen, Germany). In brief, the lyophilized and deprived of lipids hemolymph extract was reconstituted in 0.1% (v/v) trifluoroacetic acid (TFA), filtered through a 0.45 μm filter and subjected to high-performance liquid chromatography (HPLC) using a Discovery Bio Wide Pore C18 250 mm × 4.6 mm column (Sigma-Aldrich, St. Louis, MO) and two buffer sets:…”

The effect of Galleria mellonella hemolymph polypeptides on Legionella gormanii.

“…When the extract was used at the concentration 0.4 mg/ ml, ca. 50% decrease of L. dumoffii survival rate was observed (Palusinska-Szysz et al, 2012). A main protein component of G. mellonella hemolymph extract is apoLp-III (Cytryńska et al, 2007).…”

“…Antimicrobial assays. The activity of immune hemolymph extract and apoLp-III against L. gormanii was carried out using a colony counting assay as described previously (Palusinska-Szysz et al, 2012). Briefly, 10 µl of a bacterial suspension (obtained by 2×10 -4 dilution of a suspension with OD 620 = 0.1) was incubated without (control) or with the extract (final protein concentrations 0.025-0.8 mg/ml) or apoLp-III (final protein concentrations 0.025-0.2 mg/ml) at 37°C for 1 h. Then, the incubation mixtures were spread onto BCYE medium plates, incubated for 4 days at 37°C, and the number of the colony-forming units (CFU) was determined.…”

“…In our previous study, anti-L. dumoffii activity of G. mellonella defensin and apoLp-III has been documented indicating promising potential of the insect defence factors in fighting Legionella spp. (Palusinska-Szysz et al, 2012).…”