The human LL-37 peptide exerts antimicrobial activity against Legionella micdadei interacting with membrane phospholipids.

Legionella micdadei is responsible for community- or nosocomial-acquired pneumonia as well as the influenza-like illness Pontiac fever. The aim of this study was to investigate the ability of L. micdadei to utilize extracellular choline for phosphatidylcholine (PC) synthesis and its consequences for the phospholipid composition of its membrane system and the interaction with the human LL-37 peptide. Comparative analysis of the PC content using isotopic labeling revealed that in presence of exogenous choline 98% of the total PC was synthesized via the Pcs pathway while the remaining 2% were generated via the PE-methylation (PmtA) pathway. PC species were to a greater extent defined by the Pcs pathway in the outer membrane than in the inner membrane. While no major changes in the bacterial lipid content were observed using 31P NMR, indication for utilization of longer acyl chains and slight increase of PG in response to choline addition was observed by a top-down lipidomics screen. The LL-37 peptide inhibited L. micdadei growth in a dose-dependent manner. Bacteria cultured with exogenous choline were more sensitive to the LL-37 peptide when compared to the standard culture condition. Our biophysical investigations show that the peptide perturbs bacterial-derived phospholipid monolayers and this interaction is dependent on the molar portion of PC. This interaction is responsible for the observed changes in the anti-L. micdadei activity of the LL-37 peptide.

Identification of unusual phospholipid fatty acyl compositions of Acanthamoeba castellanii.

Acanthamoeba are opportunistic protozoan pathogens that may lead to sight-threatening keratitis and fatal granulomatous encephalitis. The successful prognosis requires early diagnosis and differentiation of pathogenic Acanthamoeba followed by aggressive treatment regimen. The plasma membrane of Acanthamoeba consists of 25% phospholipids (PL). The presence of C20 and, recently reported, 28- and 30-carbon fatty acyl residues is characteristic of amoeba PL. A detailed knowledge about this unusual PL composition could help to differentiate Acanthamoeba from other parasites, e.g. bacteria and develop more efficient treatment strategies. Therefore, the detailed PL composition of Acanthamoeba castellanii was investigated by 31P nuclear magnetic resonance spectroscopy, thin-layer chromatography, gas chromatography, high performance liquid chromatography and liquid chromatography-mass spectrometry. Normal and reversed phase liquid chromatography coupled with mass spectrometric detection was used for detailed characterization of the fatty acyl composition of each detected PL. The most abundant fatty acyl residues in each PL class were octadecanoyl (18∶0), octadecenoyl (18∶1 Δ9) and hexadecanoyl (16∶0). However, some selected PLs contained also very long fatty acyl chains: the presence of 28- and 30-carbon fatty acyl residues was confirmed in phosphatidylethanolamine (PE), phosphatidylserine, phosphatidic acid and cardiolipin. The majority of these fatty acyl residues were also identified in PE that resulted in the following composition: 28∶1/20∶2, 30∶2/18∶1, 28∶0/20∶2, 30∶2/20∶4 and 30∶3/20∶3. The PL of amoebae are significantly different in comparison to other cells: we describe here for the first time unusual, very long chain fatty acids with Δ5-unsaturation (30∶35,21,24) and 30∶221,24 localized exclusively in specific phospholipid classes of A. castellanii protozoa that could serve as specific biomarkers for the presence of these microorganisms.

Legionella dumoffii utilizes exogenous choline for phosphatidylcholine synthesis.

Phosphatidycholine (PC) is the major membrane-forming phospholipid in eukaryotes but it has been found in only a limited number of prokaryotes. Bacteria synthesize PC via the phospholipid N-methylation pathway (Pmt) or via the phosphatidylcholine synthase pathway (Pcs) or both. Here, we demonstrated that Legionella dumoffii has the ability to utilize exogenous choline for phosphatidylcholine (PC) synthesis when bacteria grow in the presence of choline. The Pcs seems to be a primary pathway for synthesis of this phospholipid in L. dumoffii. Structurally different PC species were distributed in the outer and inner membranes. As shown by the LC/ESI-MS analyses, PC15:0/15:0, PC16:0/15:0, and PC17:0/17:1 were identified in the outer membrane and PC14:0/16:0, PC16:0/17:1, and PC20:0/15:0 in the inner membrane. L. dumoffii pcsA gene encoding phosphatidylcholine synthase revealed the highest sequence identity to pcsA of L. bozemanae (82%) and L. longbeachae (81%) and lower identity to pcsA of L. drancourtii (78%) and L. pneumophila (71%). The level of TNF-α in THP1-differentiated cells induced by live and temperature-killed L. dumoffii cultured on a medium supplemented with choline was assessed. Live L. dumoffii bacteria cultured on the choline-supplemented medium induced TNF-α three-fold less efficiently than cells grown on the non-supplemented medium. There is an evident effect of PC modification, which impairs the macrophage inflammatory response.

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.