Susceptibility of Legionella gormanii Membrane-Derived Phospholipids to the Peptide Action of Antimicrobial LL-37-Langmuir Monolayer Studies.

LL-37 is the only member of the cathelicidin-type host defense peptide family in humans. It exhibits broad-spectrum bactericidal activity, which represents a distinctive advantage for future therapeutic targets. The presence of choline in the growth medium for bacteria changes the composition and physicochemical properties of their membranes, which affects LL-37's activity as an antimicrobial agent. In this study, the effect of the LL-37 peptide on the phospholipid monolayers at the liquid-air interface imitating the membranes of Legionella gormanii bacteria was determined. The Langmuir monolayer technique was employed to prepare model membranes composed of individual classes of phospholipids-phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL)-isolated from L. gormanii bacteria supplemented or non-supplemented with exogenous choline. Compression isotherms were obtained for the monolayers with or without the addition of the peptide to the subphase. Then, penetration tests were carried out for the phospholipid monolayers compressed to a surface pressure of 30 mN/m, followed by the insertion of the peptide into the subphase. Changes in the mean molecular area were observed over time. Our findings demonstrate the diversified effect of LL-37 on the phospholipid monolayers, depending on the bacteria growth conditions. The substantial changes in membrane properties due to its interactions with LL-37 enable us to propose a feasible mechanism of peptide action at a molecular level. This can be associated with the stable incorporation of the peptide inside the monolayer or with the disruption of the membrane leading to the removal (desorption) of molecules into the subphase. Understanding the role of antimicrobial peptides is crucial for the design and development of new strategies and routes for combating resistance to conventional antibiotics.

Pantoea trifolii sp. nov., a novel bacterium isolated from Trifolium rubens root nodules.

A novel bacterium, designated strain MMK2T, was isolated from a surface-sterilised root nodule of a Trifolium rubens plant growing in south-eastern Poland. Cells were Gram negative, non-spore forming and rod shaped. The strain had the highest 16S rRNA gene sequence similarity with P. endophytica (99.4%), P. leporis (99.4%) P. rwandensis (98.8%) and P. rodasii (98.45%). Phylogenomic analysis clearly showed that strain MMK2T and an additional strain, MMK3, should reside in the genus Pantoea and that they were most closely related to P. endophytica and P. leporis. Genome comparisons showed that the novel strain shared 82.96-93.50% average nucleotide identity and 26.2-53. 2% digital DNA:DNA hybridization with closely related species. Both strains produced siderophores and were able to solubilise phosphates. The MMK2T strain was also able to produce indole-3-acetic acid. The tested strains differed in their antimicrobial activity, but both were able to inhibit the growth of Sclerotinia sclerotiorum 10Ss01. Based on the results of the phenotypic, phylogenomic, genomic and chemotaxonomic analyses, strains MMK2T and MMK3 belong to a novel species in the genus Pantoea for which the name Pantoea trifolii sp. nov. is proposed with the type strain MMK2T (= DSM 115063T = LMG 33049T).

Potential Proallergenic Activity of Phytopathogenic Erysiphe palczewskii and Erysiphe convolvuli in in vitro Studies.

Purpose: Allergic diseases have reached epidemic proportions globally, affecting nearly 30% of the world's population. One of the most prominent sources of allergens is fungi, causing up to 6% of respiratory diseases in the general population. However, the cause of respiratory allergies is not always identifiable. Therefore, we studied the ability of two representatives of common powdery mildew (Erysiphales), Erysiphe palczewskii and Erysiphe convolvuli, to induce a proinflammatory response in in vitro models of the upper and lower respiratory tract. Materials and Methods: Two cell lines, BEAS-2B and A549, were used to mimic upper and lower respiratory epithelial cells. The toxicity of fungal extracts was assessed with MTT and flow cytometry assay. The production of reactive oxygen species in the cells was measured with flow cytometry. ELISA tests were used to determine the production of proinflammatory cytokines. The presence of the cell integrity marker was assessed with the immunofluorescence method. Results: In both cell lines, the extract of E. palczewskii and E. convolvuli microfungi induced marked production of proinflammatory IL-1ß, TNF-α, and GM-CSF cytokines involved in developing allergic reactions. The higher levels of these cytokines with higher reactive oxygen species synthesis positively correlated with the disruption of epithelial cell junctions. Conclusion: We conclude that E. palczewskii and E. convolvuli microfungi have strong proinflammatory and proallergenic potential, but this finding needs in vivo confirmation.

Lipopolysaccharide of Legionella pneumophila Serogroup 1 Facilitates Interaction with Host Cells.

Legionella pneumophila is the primary causative agent of Legionnaires' disease. The mutant-type strain interrupted in the ORF7 gene region responsible for the lipopolysaccharide biosynthesis of the L. pneumophila strain Heysham-1, lacking the O-acetyl groups attached to the rhamnose of the core part, showed a higher surface polarity compared with the wild-type strain. The measurement of excitation energy transfer between fluorophores located on the surface of bacteria and eukaryotic cells showed that, at an early stage of interaction with host cells, the mutant exhibited weaker interactions with Acanthamoeba castellanii cells and THP-1-derived macrophages. The mutant displayed reduced adherence to macrophages but enhanced adherence to A. castellanii, suggesting that the O-acetyl group of the LPS core region plays a crucial role in facilitating interaction with macrophages. The lack of core rhamnose O-acetyl groups made it easier for the bacteria to multiply in amoebae and macrophages. The mutant induced TNF-α production more strongly compared with the wild-type strain. The mutant synthesized twice as many ceramides Cer(t34:0) and Cer(t38:0) than the wild-type strain. The study showed that the internal sugars of the LPS core region of L. pneumophila sg 1 can interact with eukaryotic cell surface receptors and mediate in contacting and attaching bacteria to host cells as well as modulating the immune response to infection.

Insight into the Mechanism of Interactions between the LL-37 Peptide and Model Membranes of Legionella gormanii Bacteria.

Legionella gormanii is a fastidious, Gram-negative bacterium known to be the etiological agent of atypical community-acquired pneumonia. The human cathelicidin LL-37 exhibits a dose-dependent bactericidal effect on L. gormanii. The LL-37 peptide at the concentration of 10 µM causes the bacteria to become viable but not cultured. The antibacterial activity of the peptide is attributed to its effective binding to the bacterial membrane, as demonstrated by the fluorescence lifetime imaging microscopy. In this study, to mimic the L. gormanii membranes and their response to the antimicrobial peptide, Langmuir monolayers were used with the addition of the LL-37 peptide to the subphase of the Langmuir trough to represent the extracellular fluid. The properties of the model membranes (Langmuir monolayers) formed by phospholipids (PL) isolated from the L. gormanii bacteria cultured on the non-supplemented (PL-choline) and choline-supplemented (PL+choline) medium were determined, along with the effect of the LL-37 peptide on the intermolecular interactions, packing, and ordering under the monolayer compression. Penetration tests at the constant surface pressure were carried out to investigate the mechanism of the LL-37 peptide action on the model membranes. The peptide binds to the anionic bacterial membranes preferentially, due to its positive charge. Upon binding, the LL-37 peptide can penetrate into the hydrophobic tails of phospholipids, destabilizing membrane integrity. The above process can entail membrane disruption and ultimately cell death. The ability to evoke such a great membrane destabilization is dependent on the share of electrostatic, hydrogen bonding and Lifshitz-van der Waals LL-37-PL interactions. Thus, the LL-37 peptide action depends on the changes in the lipid membrane composition caused by the utilization of exogenous choline by the L. gormanii.

Physicochemical Characteristics of Model Membranes Composed of Legionella gormanii Lipids.

Legionella gormanii is one of the species belonging to the genus Legionella, which causes atypical community-acquired pneumonia. The most important virulence factors that enable the bacteria to colonize the host organism are associated with the cell surface. Lipids building the cell envelope are crucial not only for the membrane integrity of L. gormanii but also by virtue of being a dynamic site of interactions between the pathogen and the metabolites supplied by its host. The utilization of exogenous choline by the Legionella species results in changes in the lipids' composition, which influences the physicochemical properties of the cell surface. The aim of this study was to characterize the interfacial properties of the phospholipids extracted from L. gormanii cultured with (PL+choline) and without exogenous choline (PL-choline). The Langmuir monolayer technique coupled with the surface potential (SPOT) sensor and the Brewster angle microscope (BAM) made it possible to prepare the lipid monomolecular films (model membranes) and study their properties at the liquid/air interface at 20 °C and 37 °C. The results indicate the effect of the choline addition to the bacterial medium on the properties of the L. gormanii phospholipid membranes. The differences were revealed in the organization of monolayers, their molecular packing and ordering, degree of condensation and changes in the components' miscibility. These findings are the basis for further research on the mechanisms of adaptation of this pathogen, which by changing the native composition and properties of lipids, bypasses the action of antimicrobial compounds and avoids the host immune attack.

Unusual Lipid Components of Legionella gormanii Membranes.

Legionella spp. cause Legionnaires' disease with pneumonia as the predominant clinical symptom. L. gormanii is the second most prevalent causative agent of community-acquired pneumonia after L. pneumophila. The study aimed to characterize the lipidome of L. gormanii membranes and the importance of these analyses in bacterial chemotaxonomy. Lipidomic analyses based on ultra-high performance liquid chromatography-mass spectrometry allowed the detection of individual molecular species of a wide range of L. gormanii membrane lipids contained in the outer (OM) and inner membranes (IM). The lipid profile comprised glycerolipids (triglycerides, diglycerides), phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, cardiolipin), and sphingolipids (ceramides, hexosylceramides). The most abundant lipid fraction in the IM and OM were phospholipids. The lipidomic analysis showed that two independent phosphatidylcholine (PC) synthesis pathways operating in L. gormanii: the PE-methylation (PmtA) pathway and the PC synthase (Pcs) pathway. Comparison of the molecular profile of PC species contained in the lipids of L. gormanii membranes cultured on the medium, with and without exogenous choline, showed quantitative differences in the PC pool. An unusual feature of the L. gormanii lipids was the presence of ceramides and hexosylceramides, which are typical components of eukaryotic cells and a very small group of bacteria. To the best of our knowledge, this is the first report of the occurrence of ceramides in Legionella bacteria.

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.

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.

Fatty acid composition and lipid profiles as chemotaxonomic markers of phytopathogenic fungi Puccinia malvacearum and P. glechomatis.

The analysis of the overall fatty acid pattern as well as their distribution in various lipid classes of phytopathogenic fungi Puccinia malvacearum and P. glechomatis are considered as chemotaxonomic biomarkers. Puccinia malvacearum on Alcea rosea and P. glechomatis on Glechoma hederacea collected from plants grown in various localities were analysed to determine their fatty acid composition. Both species synthesised significant amounts of saturated palmitic and stearic acids as well as 9,10-epoxy-octadecanoic acid, which rarely occurs in the nature. Both species synthesised hydroxy FAs including 9,10-dihydroxy octadecanoic acid and long-chain 2-hydroxy fatty acids.2-hydroxy 18:0 and 3-hydroxy 20:0 fatty acids were present only in P. malvacearum spores, and these may be the chemotaxonomic markers of the species. Ultra-high performance liquid chromatography mass spectrometry was performed for a comparative lipidomic analysis of P. malvacearum and P. glechomatis. The results revealed the complexity of molecular lipid species of these fungi. P. malvacearum and P. glechomatis lipids were characterised by the presence of a high number of triglyceride (TG) species. 9,10-epoxy octadecanoic fatty acid was found in TGs. Among the many types of oxidised TGs identified in P. glechomatis lipids, the most abundant species corresponds to TG(22:5+6O_17:0_18:2). P. malvacearum and P. glechomatis produced various ceramide species with different FAs from 14 to 24 chain-length. Unusual lipids like (O-acyl)-ω-hydroxy FA 18:0/18:0 in P. glechomatis and (O-acyl)-ω-hydroxy FA 18:0/20:0 and 18:0/22:0 in P. malvacearum were detected. The analysis of the polar lipid composition showed the presence of phosphatidylcholine and phosphatidylethanolamine as the main phospholipid classes of Puccinia spp. with the highest diversity of molecular species. Other phospholipids phosphatidic acid, phosphatidylglycerol phosphatidylserine and phosphatidylinositol were present in smaller amounts. The diversity of the neutral and polar lipid composition and fatty acid profile of P. malvacearum and P. glechomatis can be used in chemotaxonomic studies.

The Role of Lipids in Legionella-Host Interaction.

Legionella are Gram-stain-negative rods associated with water environments: either natural or man-made systems. The inhalation of aerosols containing Legionella bacteria leads to the development of a severe pneumonia termed Legionnaires' disease. To establish an infection, these bacteria adapt to growth in the hostile environment of the host through the unusual structures of macromolecules that build the cell surface. The outer membrane of the cell envelope is a lipid bilayer with an asymmetric composition mostly of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. The major membrane-forming phospholipid of Legionella spp. is phosphatidylcholine (PC)-a typical eukaryotic glycerophospholipid. PC synthesis in Legionella cells occurs via two independent pathways: the N-methylation (Pmt) pathway and the Pcs pathway. The utilisation of exogenous choline by Legionella spp. leads to changes in the composition of lipids and proteins, which influences the physicochemical properties of the cell surface. This phenotypic plasticity of the Legionella cell envelope determines the mode of interaction with the macrophages, which results in a decrease in the production of proinflammatory cytokines and modulates the interaction with antimicrobial peptides and proteins. The surface-exposed O-chain of Legionella pneumophila sg1 LPS consisting of a homopolymer of 5-acetamidino-7-acetamido-8-O-acetyl-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic acid is probably the first component in contact with the host cell that anchors the bacteria in the host membrane. Unusual in terms of the structure and function of individual LPS regions, it makes an important contribution to the antigenicity and pathogenicity of Legionella bacteria.

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.

Identification and characterization of Staphylococcus spp. and their susceptibility to insect apolipophorin III.

Aim: This study investigated the effect of an insect antimicrobial protein, apolipophorin III (apoLp-III), against two newly isolated, identified and characterized clinical strains of Staphylococcus spp. Materials & methods: Both strains were identified by 16S rRNA sequencing and metabolic and phenotypic profiling. The antibacterial activity of apoLp-III was tested using a colony counting assay. ApoLp-III interaction with bacterial cell surface was analyzed by Fourier transform IR spectroscopy. Results:Staphylococcus epidermidis and Staphylococcus capitis were identified. ApoLp-III exerted a dose-dependent bactericidal effect on the tested strains. The differences in the Staphylococcus spp. surface components may contribute to the various sensitivities of these strains to apoLp-III. Conclusion: ApoLp-III may provide a baseline for development of antibacterial preparations against Staphylococcus spp. involved in dermatological problems.

Genetic diversity of Legionella pcs and pmtA genes and the effect of utilization of choline by Legionella spp. on induction of proinflammatory cytokines.

Legionella species synthesize phosphatidylcholine (PC) in two independent pathways: the three-step methylation of phosphatidylethanolamine PMT pathway and the one-step PCS pathway, in which the Pcs enzyme catalyzes the reaction between choline and CDP-diacylglycerol to form PC. Legionella pcs genes encode highly hydrophobic proteins with phosphatidylcholine synthase activity, which contain up to eight transmembrane helices with N- and C-termini located inside the bacterial cell. The comparative analysis of nucleotide sequences of pcs showed that these genes share high sequence identity among members of the Legionellaceae family. Legionella pmtA genes involved in the PMT pathway encoded small cytosolic proteins with putative phosphatidylethanolamine N-methyltransferase activity. The pmtA genes identified in Legionella species had lower sequence identity to each other than the pcs genes. The phylogenetic tree constructed based on the pcs and pmtA gene sequences showed phylogenetic relatedness between Legionella spp. and other bacteria. The utilization of extracellular choline by the four Legionella species leads to changes not only in the lipid components but also in proteins, and the interactions between these components lead to changes in cell surface properties, which result in a decline in induction of proinflammatory cytokines (TNF-α and IL-6).

The Role of Legionella pneumophila Serogroup 1 Lipopolysaccharide in Host-Pathogen Interaction.

The Legionella pneumophila TF3/1 mutant of the Corby strain, which possesses a point mutation in the active site of the O-acetyltransferase, synthesized the polysaccharide chain with a reduced degree of substitution with O-acetyl groups. The mutant did not produce a high-molecular-weight lipopolysaccharide (LPS) fraction above 12 kDa. The disturbances in LPS synthesis have an effect on the composition of other macromolecules (lipids and proteins), as indicated by differences in the infrared absorption spectra between the L. pneumophila Corby strain and its TF3/1 mutant. The wild-type strain contained less N+-CH3 and C-N groups as well as more CH3 groups than the mutant. The fatty acid composition showed that the wild type strain synthesized more branched acyl residues (a15:0, i16:0, and a17:0), a less unsaturated acid (16:1), and a straight-chain acid (18:0) than the mutant. The mutant synthesized approximately twice more a long-chain fatty acid (20:0) than the wild type. The main differences in the phospholipids between both strains were found in the classes of phosphatidylcholines and phosphatidylglycerols (PG). Substantial differences in the cell surface topography of these bacteria and their nanomechanical properties were shown by atomic force microscopy (AFM). The wild type strain had no undulated surface and produced numerous vesicles. In the case of the mutant type, the vesicles were not numerous, but there were grooves on the cell surface. The average roughness of the cell surface of the mutant was approximately twofold higher than in the wild-type strain. In turn, the wild-type strain exhibited much better adhesive properties than the mutant. The kinetic study of the interaction between the L. pneumophila strains and Acanthamoeba castellanii monitored by Förster resonance energy transfer revealed a pronounced difference, i.e., almost instantaneous and highly efficient binding of the L. pneumophila Corby strain to the amoeba surface, followed by penetration into the amoeba cells. This process was clearly not as efficient in the case of the mutant. The results point to LPS and, in particular, to the length of the polysaccharide fraction as an important L. pneumophila determinant involved in the process of adhesion to the host cell.

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.

Low temperature adaptation and the effects of cryoprotectants on mesorhizobia strains.

In this study, the tolerance of Mesorhizobium sp. ACMP18, Mesorhizobium sp. USDA3350, and Mesorhizobium temperatum LMG23931 strains, to cold and freezing were investigated. The ability to withstand freezing at -20 °C and -70 °C for 24 months was different among the studied strains and depended on the cryoprotectant used. The survivability of mesorhizobial strains at -20 °C and -70 °C was significantly improved by some cryoprotectans (glycerol and sucrose/peptone). It is worth noting that the greatest resistance to freezing was detected when stress treatments were performed in glycerol as a cryoprotectant. Using PCR analysis, cspA genes were identified in the studied strains. Their nucleotide sequences were most similar to the sequences of the corresponding genes of the Mesorhizobium species. The expression of the cspA gene in the studied bacteria was analyzed using the RT-PCR technique. The fatty acid composition of the mesorhizobia was determined at 5, 10, 15, and 28 °C. It was noticed that growth temperature significantly affected the fatty acid composition and the amounts of unsaturated fatty acids, especially that of cis-vaccenic acid (18:1É·(11)), increased markedly in bacterial cells cultivated at 5, 10, and 15 °C.

PCR method for the rapid detection and discrimination of Legionella spp. based on the amplification of pcs, pmtA, and 16S rRNA genes.

Legionella bacteria are organisms of public health interest due to their ability to cause pneumonia (Legionnaires' disease) in susceptible humans and their ubiquitous presence in water supply systems. Rapid diagnosis of Legionnaires' disease allows the use of therapy specific for the disease. L. pneumophila serogroup 1 is the most common cause of infection acquired in community and hospital environments. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this work, simplex and duplex PCR assays with the use of new molecular markers pcs and pmtA involved in phosphatidylcholine synthesis were specified for rapid and cost-efficient identification and distinguishing Legionella species. The sets of primers developed were found to be sensitive and specific for reliable detection of Legionella belonging to the eight most clinically relevant species. Among these, four primer sets I, II, VI, and VII used for duplex-PCRs proved to have the highest identification power and reliability in the detection of the bacteria. Application of this PCR-based method should improve detection of Legionella spp. in both clinical and environmental settings and facilitate molecular typing of these organisms.

Analysis of cell surface alterations in Legionella pneumophila cells treated with human apolipoprotein E.

Binding of human apolipoprotein E (apoE) to Legionella pneumophila lipopolysaccharide was analysed at the molecular level by Fourier-transform infrared spectroscopy, thereby providing biophysical evidence for apoE-L. pneumophila lipopolysaccharide interaction. Atomic force microscopy imaging of apoE-exposed L. pneumophila cells revealed alterations in the bacterial cell surface topography and nanomechanical properties in comparison with control bacteria. The changes induced by apoE binding to lipopolysaccharide on the surface of L. pneumophila cells may participate in: (1) impeding the penetration of host cells by the bacteria; (2) suppression of pathogen intracellular growth and eventually; and (3) inhibition of the development of infection.