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.

BL-11C Micro-MX: a high-flux microfocus macromolecular-crystallography beamline for micrometre-sized protein crystals at Pohang Light Source II.

BL-11C, a new protein crystallography beamline, is an in-vacuum undulator-based microfocus beamline used for macromolecular crystallography at the Pohang Accelerator Laboratory and it was made available to users in June 2017. The beamline is energy tunable in the range 5.0-20 keV to support conventional single- and multi-wavelength anomalous-dispersion experiments against a wide range of heavy metals. At the standard working energy of 12.659 keV, the monochromated beam is focused to 4.1 µm (V) × 8.5 µm (H) full width at half-maximum at the sample position and the measured photon flux is 1.3 × 1012 photons s-1. The experimental station is equipped with a Pilatus3 6M detector, a micro-diffractometer (MD2S) incorporating a multi-axis goniometer, and a robotic sample exchanger (CATS) with a dewar capacity of 90 samples. This beamline is suitable for structural determination of weakly diffracting crystalline substances, such as biomaterials, including protein, nucleic acids and their complexes. In addition, serial crystallography experiments for determining crystal structures at room temperature are possible. Herein, the current beamline characteristics, technical information for users and some recent scientific highlights are described.

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.

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).

Functional Analysis of the Alternative Sigma-28 Factor FliA and Its Anti-Sigma Factor FlgM of the Nonflagellated Legionella Species L. oakridgensis.

Legionella oakridgensis causes Legionnaires' disease but is known to be less virulent than Legionella pneumophilaL. oakridgensis is one of the Legionella species that is nonflagellated. The genes of the flagellar regulon are absent, except those encoding the alternative sigma-28 factor (FliA) and its anti-sigma-28 factor (FlgM). Similar to L. oakridgensis, Legionella adelaidensis and Legionella londiniensis, located in the same phylogenetic clade, have no flagellar regulon, although both are positive for fliA and flgM Here, we investigated the role and function of both genes to better understand the role of FliA, the positive regulator of flagellin expression, in nonflagellated strains. We demonstrated that the FliA gene of L. oakridgensis encodes a functional sigma-28 factor that enables the transcription start from the sigma-28-dependent promoter site. The investigations have shown that FliA is necessary for full fitness of L. oakridgensis Interestingly, expression of FliA-dependent genes depends on the growth phase and temperature, as already shown for L. pneumophila strains that are flagellated. In addition, we demonstrated that FlgM is a negative regulator of FliA-dependent gene expression. FlgM seems to be degraded in a growth-phase- and temperature-dependent manner, instead of being exported into the medium as reported for most bacteria. The degradation of FlgM leads to an increase of FliA activity.IMPORTANCE A less virulent Legionella species, L. oakridgensis, causes Legionnaires' disease and is known to not have flagella, even though L. oakridgensis has the regulator of flagellin expression (FliA). This protein has been shown to be involved in the expression of virulence factors. Thus, the strain was chosen for use in this investigation to search for FliA target genes and to identify putative virulence factors of L. oakridgensis One of the five major target genes of FliA identified here encodes the anti-FliA sigma factor FlgM. Interestingly, in contrast to most homologs in other bacteria, FlgM in L. oakridgensis seems not to be transported from the cell so that FliA gets activated. In L. oakridgensis, FlgM seems to be degraded by protease activities.

Legionella clemsonensis sp. nov.: a green fluorescing Legionella strain from a patient with pneumonia.

A novel Legionella species was identified based on sequencing, cellular fatty acid analysis, biochemical reactions, and biofilm characterization. Strain D5610 was originally isolated from the bronchial wash of a patient in Ohio, USA. The bacteria were gram-negative, rod-shaped, and exhibited green fluorescence under long wave UV light. Phylogenetic analysis and fatty acid composition revealed a distinct separation within the genus. The strain grows between 26-45°C and forms biofilms equivalent to L. pneumophila Philadelphia 1. These characteristics suggest that this isolate is a novel Legionella species, for which the name Legionella clemsonensis sp nov. is proposed.

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.

Legionella thermalis sp. nov., isolated from hot spring water in Tokyo, Japan.

Strain L-47(T) of a novel bacterial species belonging to the genus Legionella was isolated from a sample of hot spring water from Tokyo, Japan. The 16S rRNA gene sequences (1477 bp) of this strain (accession number AB899895) had less than 95.0% identity with other Legionella species. The dominant fatty acids of strain L-47(T) were a15:0 (29.6%) and the major ubiquinone was Q-12 (71.1%). It had a guanine-plus-cytosine content of 41.5 mol%. The taxonomic description of Legionella thermalis sp. nov. is proposed to be type strain L-47(T) (JCM 30970(T) = KCTC 42799(T)).

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.

Characterization of legionella lipopolysaccharide.

The lipopolysaccharide(LPS) of Legionella spp. is an immuno-dominant antigen and the basis for Legionella pneumophila serogroup classification. The LPS shows a peculiar structure composed of a very hydrophobic lipid A acylated by long chain fatty acids and an O-antigen-specific chain consisting of homopolymeric legionaminic acid. In this chapter we describe a method for the isolation of LPS from L. pneumophila. In the first part we describe the chemical purification, in the second part we outline the application of monoclonal antibody (mAb) in Western blot and immuno-localization by indirect immunofluorescence. This report does not describe physico-chemical methods that analyze the structure of lipopolysaccharide entities.

Secretive bacterial pathogens and the secretory pathway.

Eukaryotic cells possess two extensive endomembrane systems, each consisting of several sub-compartments connected by vesicular trafficking. One of these systems, the endocytic pathway, serves incoming traffic, and the other system, the secretory pathway (SP), is responsible for surface-bound traffic of intracellularly formed vesicles. Compartments derived of either system can be colonized by intracellular pathogens. In this review, we discuss the interactions between the SP and prominent intracellular bacterial pathogens of the genera Legionella, Brucella, Chlamydia and Salmonella. We emphasize secreted bacterial effector proteins, which directly manipulate host components of this pathway.

Rapid identification of Legionella spp. by MALDI-TOF MS based protein mass fingerprinting.

A set of reference strains representing 38 different Legionella species were submitted to Whole Cell Mass Spectrometry (WCMS) with MALDI-TOF. The dendrogram computed from strain mass spectral patterns obtained by WCMS was compared to the phylogenetic tree obtained from macrophage infectivity potentiator (mip) sequences. The trees inferred from these two methods revealed significant homologies. Using 453 Legionella isolates previously characterized by genotyping, it was possible to create species-specific SuperSpectra, using appropriate sets of spectral masses, allowing unambiguous differentiation and identification of the most frequently isolated Legionella species. These SuperSpectra were tested for their suitability to identify Legionella strains isolated from water samples, cooling towers, potting soils and patient specimens deposited at the Swiss National Reference Centre for Legionella and previously identified by molecular methods such as mip gene sequencing. 99.1% of the tested strains isolated from the environment could be correctly identified by comparison with the new SuperSpectra. The identification of Legionella spp. by MALDI-TOF MS is rapid, easy to perform and has the advantage of being time- and cost-saving, in comparison to sequence-based identification.

Legionella bozemanae synthesizes phosphatidylcholine from exogenous choline.

The phospholipid class and fatty acid composition of Legionella bozemanae were determined using thin-layer chromatography, gas-liquid chromatography, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were the predominant phospholipids, while phosphatidyl-N-monomethylethanolamine, phosphatidylglycerol, and phosphatidyl-N,N-dimethylethanolamine were present at low concentrations. With the use of the LC/MS technique, PC16:0/15:0, PC17:/15:0, and PE16:1/15:0 were shown to be the dominant phospholipid constituents, which may be taxonomically significant. Two independent phosphatidylcholine synthesis pathways (the three-step methylation and the one-step CDP-choline pathway) were present and functional in L. bozemanae. In the genome of L. bozemanae, genes encoding two potential phosphatidylcholine forming enzymes, phospholipid N-methyl transferase (PmtA) and phosphatidylcholine synthase (Pcs), homologous to L. longbeachae, L. drancourtii, and L. pneumophila pmtA and pcs genes were identified. Genes pmtA and pcs from L. bozemanae were sequenced and analyzed on nucleotide and amino acid levels. Bacteria grown on an artificial medium with labelled choline synthesized phosphatidylcholine predominantly via the phosphatidylcholine synthase pathway, which indicates that L. bozemanae phosphatidylcholine, similarly as in other bacteria associated with eukaryotes, is an important determinant of host-microbe interactions.

A quick and easy method to identify bacteria by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

Concerns with water quality have increased in recent years, in part due to the more frequent contamination of water by pathogens like E. coli and L. pneumophila. Current methods for the typing of bacteria in water samples are based on culture of samples on specific media. These techniques are time-consuming, subject to the impact of interferents and do not totally meet all the requirements of prevention. There is a need for accurate and rapid identification of these microorganisms. This report deals with the detection of bacteria, more precisely of Legionella spp., and the development of an analytical strategy for a rapid and unambiguous identification of these pathogens in water from diverse origins. Therefore, a protein mass mapping using matrix-assisted laser desorption/ionisation mass spectrometry (MALDI MS) of whole bacteria combined with a home-made database of bacteria spectra is applied. A large variety of different bacteria and microorganisms is used to approach the actual composition of samples with numerous interferents. The objective is to propose a universal method for sampling preparation before MALDI MS analysis and optimised spectrometric conditions for reproducible intense peaks. Several experimental factors known to influence signal quality such as time and media of culture have been studied. The proposed method gives promising results for a sure differentiation of Legionella species and subspecies and a rapid identification of bacteria which are the most dangerous or difficult to eradicate. This method is easy to perform with an excellent reproducibility. The analytical protocol and the corresponding database were validated on samples from different origins (cooling tower, plumbing hot water).

Rapid identification of Legionella species by mass spectrometry.

Legionella species are facultative, intracellular bacteria that infect macrophages and protozoa, with the latter acting as transmission vectors to humans. These fastidious bacteria mostly cause pulmonary tract infections and are routinely identified by various molecular methods, mainly PCR targeting the mip gene and sequencing, which are expensive and time-consuming. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has emerged as a rapid and inexpensive method for identification of bacterial species. This study evaluated the use of MALDI-TOF-MS for rapid species and serogroup identification of 21 Legionella species recognized as human pathogens. To this end, a reference MS database was developed including 59 Legionella type strains, and a blind test was performed using 237 strains from various species. Two hundred and twenty-three of the 237 strains (94.1 %) were correctly identified at the species level, although ten (4.2 %) were identified with a score lower than 2.0. Fourteen strains (5.9 %) from eight species were misidentified at the species level, including seven (3.0 %) with a significant score, suggesting an intraspecific variability of protein profiles within some species. MALDI-TOF-MS was reproducible but could not identify Legionella strains at the serogroup level. When compared with mip gene sequencing, MALDI-TOF-MS exhibited a sensitivity of 99.2 and 89.9 % for the identification of Legionella strains at the genus and species level, respectively. This study demonstrated that MALDI-TOF-MS is a reliable tool for the rapid identification of Legionella strains at the species level.

Cellular envelope phospholipids from Legionella lytica.

The composition of phospholipids from the cellular envelope of Legionella lytica grown on artificial medium was determined by two-dimensional thin-layer chromatography. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidyl-N-monomethylethanolamine were the predominant phospholipids, while diphosphatidylglycerol, phosphatidylglycerol, and phosphatidyl-N,N-dimethylethanolamine were present at low concentrations. A trace amount of lipids carrying glycosyl residues was also observed. The fatty acids and their distribution in individual phospholipids were characterized using liquid chromatography/mass spectrometry (LC/MS), matrix-assisted laser desorption ionization-time of flight, and gas chromatography/MS methods. The characteristic feature of L. lytica phospholipids was the presence of an unbranched chain (which differentiates this bacterium from Legionella pneumophila) and branched iso and anteiso fatty acids as well as cis-9,10-methylenehexadecanoic acid. According to spectroscopic LC/MS data, the localization of saturated and unsaturated fatty acid residues on phosphorylglycerol was determined. Some aspects of the significance of phosphatidylcholine, one of the main phospholipids in L. lytica, are addressed and taxonomic implications of the data are discussed.

Complete nucleotide sequence of pLD-TEX-KL, a 66-kb plasmid of Legionella dumoffii TEX-KL strain.

The complete nucleotide sequence of a large (66 kb) plasmid pLD-TEX-KL of Legionella dumoffii TEX-KL strain was determined. Of the 57 predicted open reading frames (ORFs), 39 (68%) encoded proteins similar to previously known proteins, five (9%) were assigned with putative functions, three (5%) encoded conserved hypothetical proteins, and 10 (18%) had no homology to any genes present in the current open databases. The ORFs with similar functions were organized in a modular structure; thus, transfer region was identified, as well as a putative heavy-metal ion transporter system (hel). The transfer region encoded homologs of the Salmonella entrica serovar Typhi conjugative system components involved in conjugation. In addition, we also found a potential protein that was analogous to the DNA polymerase III epsilon subunit. It is rarely found that plasmid encode the DNA polymerase.

The HopX (AvrPphE) family of Pseudomonas syringae type III effectors require a catalytic triad and a novel N-terminal domain for function.

Many gram-negative plant pathogenic bacteria employ type III secretion systems to deliver effector proteins directly into the host cell during infection. On susceptible hosts, type III effectors aid pathogen growth by manipulating host defense pathways. On resistant hosts, some effectors can activate specific host disease resistance (R) genes, leading to generation of rapid and effective immune responses. The biochemical basis of these processes is poorly understood. The HopX (AvrPphE) family is a widespread type III effector among phytopathogenic bacteria. We determined that HopX family members are modular proteins composed of a conserved putative cysteine-based catalytic triad and a conserved potential target/cofactor interaction domain. HopX is soluble in host cells. Putative catalytic triad residues are required for avirulence activity on resistant bean hosts and for the generation of a cell-death response in specific Arabidopsis genotypes. The putative target/cofactor interaction domain is also required for these activities. Our data suggest that specific interaction with and modification of a cytosolic host target drives HopX recognition in resistant hosts and may contribute to virulence in susceptible hosts. Surprisingly, the Legionella pneumophila genome was found to contain a protein with similarity to HopX in sequence and domain arrangement, suggesting that these proteins might also contribute to animal pathogenesis and could be delivered to plant and animal hosts by diverse secretion systems.

[Physicochemical factors influencing distribution of Legionella species in Japanese hot springs].

We examined the relationship between the distribution of Legionella bacteria and various physicochemical characteristics of hot springs in Japan. Legionella bacteria were isolated from 52 (49.5%) out of 105 water samples, particularly from outdoor hot springs (67.3%). The bacterial count in the water samples positive for Legionella (86.5%) ranged from 10(1) to < 10(3) cfu/100 mL. L. pneumophila serogroup (SG) 4 (27.8%) was predominant in the water samples, followed by SG 5 (12.2%). The pulsefield gel electrophoresis (PFGE) patterns of chromosomal DNA for L. pneumophila SG 4 isolated from different parts of a hot spring resort were identical. Isolation of Legionella species from hot spring waters did not occur at pH 1.8-3.3, SO4(2-): > 780 mg/L, and H2SiO3: > 146 mg/L. The hot water-recirculating systems were applied to 18 out of 20 (90%) hot spring facilities which were found positive for Legionella. These results indicate that Legionella species are widespread in hot springs throughout Japan, except for waters with a low pH and non-recirculating waters, and that a single strain of L. pneumophila SG 4 is predominant in a particular hot spring resort.