The effect of Galleria mellonella hemolymph polypeptides on Legionella gormanii

Among Legionella species, which are recognized to be pathogenic for humans, L. gormanii is the second prevalent causative agent of community-acquired pneumonia after L. pneumophila. Anti-L. gormanii activity of Galleria mellonella hemolymph extract and apolipophorin III (apoLp-III) was examined. The extract and apoLp-III at the concentration 0.025 mg/ml caused 75% and 10% decrease of the bacteria survival rate, respectively. The apoLp-III-induced changes of the bacteria cell surface were analyzed for the first time by atomic force microscopy. Our studies demonstrated the powerful anti-Legionella effects of the insect defence polypeptides, which could be exploited in drugs design against these pathogens

Anti-Legionella dumoffii activity of Galleria mellonella defensin and apolipophorin III

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

Occurrence of New Polyenoic Very Long Chain Acyl Residues in Lipids from Acanthamoeba castellanii

The cellular fatty acid composition of Acanthamoeba castellanii, a unicellular bacteriovorous organism, was reinvestigated. Lipids from amoebae grown axenically in proteose peptone-yeast extract-glucose medium were extracted with chloroform–methanol and separated by silicic acid column chromatography into non-polar and polar fractions. The fatty acid composition of the lipids and the double-bond position of the unsaturated acids have been determined by capillary gas chromatography-mass spectrometry (GC-MS) of their corresponding methyl esters, 2-alkenyl-4,4-dimethyloxazoline (DMOX) derivatives and dimethyldisulfide (DMDS) adducts. Evidence is given that lipids from A. castellanii in addition to the three already identified saturated straight chain fatty acids: tetradecanoic (C14:0), hexadecanoic (C16:0), octadecanoic (C18:0), and six preponderant unsaturated fatty acids: hexadecenoic (C16:1 Δ7), octadecenoic (C18:1 Δ9), octadecadienoic (C18:2 Δ9,12), eicosadienoic (C20:2 Δ11,14), eicosatrienoic (C20:3 Δ8,11,14), and eicosatetraenoic (C20:4 Δ5,8,11,14), contain additionally four very long chain unsaturated fatty acids: octacosenoic (C28:1 Δ21), octacosadienoic (C28:2 Δ5,21), triacontadienoic (30:2 Δ21,24), and triacontatrienoic (C30:3 Δ5,21,24) previously unreported in lipids of A. castellanii. These new long chain fatty acids account for approximately 25% of total fatty acids. To our knowledge, this is the first report of very long chain polyenoic fatty acids present in lipids extracted from A. castellanii cells

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

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

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

The Influence of Polysaccharides/TiO₂ on the Model Membranes of Dipalmitoylphosphatidylglycerol and Bacterial Lipids

The aim of the study was to determine the bactericidal properties of popular medical, pharmaceutical, and cosmetic ingredients, namely chitosan (Ch), hyaluronic acid (HA), and titanium dioxide (TiO2). The characteristics presented in this paper are based on the Langmuir monolayer studies of the model biological membranes formed on subphases with these compounds or their mixtures. To prepare the Langmuir film, 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DPPG) phospholipid, which is the component of most bacterial membranes, as well as biological material-lipids isolated from bacteria Escherichia coli and Staphylococcus aureus were used. The analysis of the surface pressure-mean molecular area (π-A) isotherms, compression modulus as a function of surface pressure, CS−1 = f(π), relative surface pressure as a function of time, π/π0 = f(t), hysteresis loops, as well as structure visualized using a Brewster angle microscope (BAM) shows clearly that Ch, HA, and TiO2 have antibacterial properties. Ch and TiO2 mostly affect S. aureus monolayer structure during compression. They can enhance the permeability of biological membranes leading to the bacteria cell death. In turn, HA has a greater impact on the thickness of E. coli film

Influence of the Antimicrobial LL-37 Peptide on <i>Legionella dumoffii</i> Phospholipids Adsorbed at the Air–Liquid Interface

Legionella dumoffii is an intracellular pathogen of freshwater protozoans capable of infecting and multiplying in mammalian cells, causing a severe respiratory disease called Legionnaires’ disease. The pathomechanism of infection development is very complex and depends on many factors, including the structure and properties of macromolecules that build the components of the L. dumoffii cell envelope. Phospholipids (PLs) forming biological membranes have a significant impact on the integrity of the membrane as well as on the interactions with the host cells. L. dumoffii changes its lipid profile under the influence of external factors, which allows it to adapt to the living environment. One of the factors altering the PL composition is the presence of exogenous choline. The aim of this study was to determine the physicochemical properties of the model bacterial membranes adsorbed at the air–liquid interface (Langmuir monolayers). They were composed of phospholipids isolated from L. dumoffii cultured with (PL+choline) and without (PL−choline) choline. Moreover, the effect of the human cathelicidin (LL-37 peptide) added to the subphase on these monolayers was analyzed in terms of phospholipid–peptide interactions. The results indicated that the monolayers of PL+choline were slightly more condensed than PL−choline. In the presence of LL-37, the elasticity of both monolayers increased; thus, their molecular packing and ordering decreased. The disturbing effect was related to the peptide’s antibacterial activity

Occurrence of New Polyenoic Very Long Chain Acyl Residues in Lipids from Acanthamoeba castellanii

The cellular fatty acid composition of Acanthamoeba castellanii, a unicellular bacteriovorous organism, was reinvestigated. Lipids from amoebae grown axenically in proteose peptone-yeast extract-glucose medium were extracted with chloroform–methanol and separated by silicic acid column chromatography into non-polar and polar fractions. The fatty acid composition of the lipids and the double-bond position of the unsaturated acids have been determined by capillary gas chromatography-mass spectrometry (GC-MS) of their corresponding methyl esters, 2-alkenyl-4,4-dimethyloxazoline (DMOX) derivatives and dimethyldisulfide (DMDS) adducts. Evidence is given that lipids from A. castellanii in addition to the three already identified saturated straight chain fatty acids: tetradecanoic (C14:0), hexadecanoic (C16:0), octadecanoic (C18:0), and six preponderant unsaturated fatty acids: hexadecenoic (C16:1 Δ7), octadecenoic (C18:1 Δ9), octadecadienoic (C18:2 Δ9,12), eicosadienoic (C20:2 Δ11,14), eicosatrienoic (C20:3 Δ8,11,14), and eicosatetraenoic (C20:4 Δ5,8,11,14), contain additionally four very long chain unsaturated fatty acids: octacosenoic (C28:1 Δ21), octacosadienoic (C28:2 Δ5,21), triacontadienoic (30:2 Δ21,24), and triacontatrienoic (C30:3 Δ5,21,24) previously unreported in lipids of A. castellanii. These new long chain fatty acids account for approximately 25% of total fatty acids. To our knowledge, this is the first report of very long chain polyenoic fatty acids present in lipids extracted from A. castellanii cells

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

AbstractThe 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