Phosphorylation of muramyl peptides by NAGK is required for NOD2 activation.

Bacterial cell wall components provide various unique molecular structures that are detected by pattern recognition receptors (PRRs) of the innate immune system as non-self. Most bacterial species form a cell wall that consists of peptidoglycan (PGN), a polymeric structure comprising alternating amino sugars that form strands cross-linked by short peptides. Muramyl dipeptide (MDP) has been well documented as a minimal immunogenic component of peptidoglycan1-3. MDP is sensed by the cytosolic nucleotide-binding oligomerization domain-containing protein 24 (NOD2). Upon engagement, it triggers pro-inflammatory gene expression, and this functionality is of critical importance in maintaining a healthy intestinal barrier function5. Here, using a forward genetic screen to identify factors required for MDP detection, we identified N-acetylglucosamine kinase (NAGK) as being essential for the immunostimulatory activity of MDP. NAGK is broadly expressed in immune cells and has previously been described to contribute to the hexosamine biosynthetic salvage pathway6. Mechanistically, NAGK functions upstream of NOD2 by directly phosphorylating the N-acetylmuramic acid moiety of MDP at the hydroxyl group of its C6 position, yielding 6-O-phospho-MDP. NAGK-phosphorylated MDP-but not unmodified MDP-constitutes an agonist for NOD2. Macrophages from mice deficient in NAGK are completely deficient in MDP sensing. These results reveal a link between amino sugar metabolism and innate immunity to bacterial cell walls.

A lectin S-domain receptor kinase mediates lipopolysaccharide sensing in Arabidopsis thaliana.

The sensing of microbe-associated molecular patterns (MAMPs) triggers innate immunity in animals and plants. Lipopolysaccharide (LPS) from Gram-negative bacteria is a potent MAMP for mammals, with the lipid A moiety activating proinflammatory responses via Toll-like receptor 4 (TLR4). Here we found that the plant Arabidopsis thaliana specifically sensed LPS of Pseudomonas and Xanthomonas. We isolated LPS-insensitive mutants defective in the bulb-type lectin S-domain-1 receptor-like kinase LORE (SD1-29), which were hypersusceptible to infection with Pseudomonas syringae. Targeted chemical degradation of LPS from Pseudomonas species suggested that LORE detected mainly the lipid A moiety of LPS. LORE conferred sensitivity to LPS onto tobacco after transient expression, which demonstrated a key function in LPS sensing and indicated the possibility of engineering resistance to bacteria in crop species.

Competence remodels the pneumococcal cell wall exposing key surface virulence factors that mediate increased host adherence.

Competence development in the human pathogen Streptococcus pneumoniae controls several features such as genetic transformation, biofilm formation, and virulence. Competent bacteria produce so-called "fratricins" such as CbpD that kill noncompetent siblings by cleaving peptidoglycan (PGN). CbpD is a choline-binding protein (CBP) that binds to phosphorylcholine residues found on wall and lipoteichoic acids (WTA and LTA) that together with PGN are major constituents of the pneumococcal cell wall. Competent pneumococci are protected against fratricide by producing the immunity protein ComM. How competence and fratricide contribute to virulence is unknown. Here, using a genome-wide CRISPRi-seq screen, we show that genes involved in teichoic acid (TA) biosynthesis are essential during competence. We demonstrate that LytR is the major enzyme mediating the final step in WTA formation, and that, together with ComM, is essential for immunity against CbpD. Importantly, we show that key virulence factors PspA and PspC become more surface-exposed at midcell during competence, in a CbpD-dependent manner. Together, our work supports a model in which activation of competence is crucial for host adherence by increased surface exposure of its various CBPs.

Lipoteichoic acids influence cell shape and bacterial division of Streptococcus suis serotype 2, but play a limited role in the pathogenesis of the infection.

Streptococcus suis serotype 2 is a major swine pathogen and a zoonotic agent, causing meningitis in both swine and humans, responsible for substantial economic losses to the swine industry worldwide. The pathogenesis of infection and the role of bacterial cell wall components in virulence have not been fully elucidated. Lipoproteins, peptidoglycan, as well as lipoteichoic acids (LTA) have all been proposed to contribute to virulence. In the present study, the role of the LTA in the pathogenesis of the infection was evaluated through the characterisation of a mutant of the S. suis serotype 2 strain P1/7 lacking the LtaS enzyme, which mediates the polymerization of the LTA poly-glycerolphosphate chain. The ltaS mutant was confirmed to completely lack LTA and displayed significant morphological defects. Although the bacterial growth of this mutant was not affected, further results showed that LTA is involved in maintaining S. suis bacterial fitness. However, its role in the pathogenesis of the infection appears limited. Indeed, LTA presence reduces self-agglutination, biofilm formation and even dendritic cell activation, which are important aspects of the pathogenesis of the infection caused by S. suis. In addition, it does not seem to play a critical role in virulence using a systemic mouse model of infection.

Prdx4 limits caspase-1 activation and restricts inflammasome-mediated signaling by extracellular vesicles.

Inflammasomes are cytosolic protein complexes, which orchestrate the maturation of active IL-1ß by proteolytic cleavage via caspase-1. Although many principles of inflammasome activation have been described, mechanisms that limit inflammasome-dependent immune responses remain poorly defined. Here, we show that the thiol-specific peroxidase peroxiredoxin-4 (Prdx4) directly regulates IL-1ß generation by interfering with caspase-1 activity. We demonstrate that caspase-1 and Prdx4 form a redox-sensitive regulatory complex via caspase-1 cysteine 397 that leads to caspase-1 sequestration and inactivation. Mice lacking Prdx4 show an increased susceptibility to LPS-induced septic shock. This effect was phenocopied in mice carrying a conditional deletion of Prdx4 in the myeloid lineage (Prdx4-ΔLysMCre). Strikingly, we demonstrate that Prdx4 co-localizes with inflammasome components in extracellular vesicles (EVs) from inflammasome-activated macrophages. Purified EVs are able to transmit a robust IL-1ß-dependent inflammatory response in vitro and also in recipient mice in vivo. Loss of Prdx4 boosts the pro-inflammatory potential of EVs. These findings identify Prdx4 as a critical regulator of inflammasome activity and provide new insights into remote cell-to-cell communication function of inflammasomes via macrophage-derived EVs.

Longitudinal gut microbiome analyses and blooms of pathogenic strains during lupus disease flares.

OBJECTIVE: Whereas genetic susceptibility for systemic lupus erythematosus (SLE) has been well explored, the triggers for clinical disease flares remain elusive. To investigate relationships between microbiota community resilience and disease activity, we performed the first longitudinal analyses of lupus gut-microbiota communities. METHODS: In an observational study, taxononomic analyses, including multivariate analysis of ß-diversity, assessed time-dependent alterations in faecal communities from patients and healthy controls. From gut blooms, strains were isolated, with genomes and associated glycans analysed. RESULTS: Multivariate analyses documented that, unlike healthy controls, significant temporal community-wide ecological microbiota instability was common in SLE patients, and transient intestinal growth spikes of several pathogenic species were documented. Expansions of only the anaerobic commensal, Ruminococcus (blautia) gnavus (RG) occurred at times of high-disease activity, and were detected in almost half of patients during lupus nephritis (LN) disease flares. Whole genome sequence analysis of RG strains isolated during these flares documented 34 genes postulated to aid adaptation and expansion within a host with an inflammatory condition. Yet, the most specific feature of strains found during lupus flares was the common expression of a novel type of cell membrane-associated lipoglycan. These lipoglycans share conserved structural features documented by mass spectroscopy, and highly immunogenic repetitive antigenic-determinants, recognised by high-level serum IgG2 antibodies, that spontaneously arose, concurrent with RG blooms and lupus flares. CONCLUSIONS: Our findings rationalise how blooms of the RG pathobiont may be common drivers of clinical flares of often remitting-relapsing lupus disease, and highlight the potential pathogenic properties of specific strains isolated from active LN patients.

Polar lipid characterization and description of Chryseobacterium capnotolerans sp. nov., isolated from high CO2-containing atmosphere and emended descriptions of the genus Chryseobacterium, and the species C. balustinum, C. daecheongense, C. formosense, C. gleum, C. indologenes, C. joostei, C. scophthalmum and C. ureilyticum.

Modified atmosphere (MA) packaging plays an important role in improving food quality and safety. By using different gas mixtures and packaging materials the shelf life of fresh produce can significantly be increased. A Gram-negative-staining, rod-shaped, orange-pigmented strain DH-B6T, has been isolated from MA packed raw pork sausage (20% CO2, 80% O2). The strain produced biofilms and showed growth at high CO2 levels of up to 40%. Complete 16S rRNA gene and whole-genome sequences revealed that strain DH-B6T belongs to the genus Chryseobacterium, being closely related to strain Chryseobacterium indologenes DSM 16777T (98.4%), followed by Chryseobacterium gleum NCTC11432T (98.3%) and Chryseobacterium lactis KC1864T (98.2%). Average nucleotide identity value between DH-B6T and C. indologenes DSM 16777T was 81.1% and digital DNA-DNA hybridisation was 24.9%, respectively. The DNA G+C content was 35.51 mol%. Chemotaxonomical analysis revealed the presence of the rare glycine lipid cytolipin, the serine-glycine lipid flavolipin and the sulfonolipid sulfobacin A, as well as phosphatidylethanolamine, monohexosyldiacylglycerol and ornithine lipid, including the hydroxylated forms. Major fatty acids were iC15 : 0 (50.7%) and iC17 : 1 cis 9 (28.7%), followed by iC15 : 0 2-OH (7.0%) and iC17 : 0 3-OH (6.2%). The isolated strain contained MK-6 as the only respiratory quinone and flexirubin-like pigments were detected as the major pigments. Based on the phenotypic, chemotaxonomic and phylogenetic characteristics, the strain DH-B6T (=DSM 110542T=LMG 31915T) represents a novel species of the genus Chryseobacterium, for which the name Chryseobacterium capnotolerans sp. nov. is proposed. Emended descriptions of the genus Chryseobacterium and eight species of this genus based on polar lipid characterisation are also proposed.

Remodeling of Lipid A in Pseudomonas syringae pv. phaseolicola In Vitro.

Pseudomonas species infect a variety of organisms, including mammals and plants. Mammalian pathogens of the Pseudomonas family modify their lipid A during host entry to evade immune responses and to create an effective barrier against different environments, for example by removal of primary acyl chains, addition of phosphoethanolamine (P-EtN) to primary phosphates, and hydroxylation of secondary acyl chains. For Pseudomonas syringae pv. phaseolicola (Pph) 1448A, an economically important pathogen of beans, we observed similar lipid A modifications by mass spectrometric analysis. Therefore, we investigated predicted proteomes of various plant-associated Pseudomonas spp. for putative lipid A-modifying proteins using the well-studied mammalian pathogen Pseudomonas aeruginosa as a reference. We generated isogenic mutant strains of candidate genes and analyzed their lipid A. We show that the function of PagL, LpxO, and EptA is generally conserved in Pph 1448A. PagL-mediated de-acylation occurs at the distal glucosamine, whereas LpxO hydroxylates the secondary acyl chain on the distal glucosamine. The addition of P-EtN catalyzed by EptA occurs at both phosphates of lipid A. Our study characterizes lipid A modifications in vitro and provides a useful set of mutant strains relevant for further functional studies on lipid A modifications in Pph 1448A.

Commensal Streptococcus mitis produces two different lipoteichoic acids of type I and type IV.

The opportunistic pathogen Streptococcus mitis possesses, like other members of the Mitis group of viridans streptococci, phosphorylcholine (P-Cho)-containing teichoic acids (TAs) in its cell wall. Bioinformatic analyses predicted the presence of TAs that are almost identical with those identified in the pathogen Streptococcus pneumoniae, but a detailed analysis of S. mitis lipoteichoic acid (LTA) was not performed to date. Here, we determined the structures of LTA from two S. mitis strains, the high-level beta-lactam and multiple antibiotic resistant strain B6 and the penicillin-sensitive strain NCTC10712. In agreement with bioinformatic predictions, we found that the structure of one LTA (type IV) was like pneumococcal LTA, except the exchange of a glucose moiety with a galactose within the repeating units. Further genome comparisons suggested that the majority of S. mitis strains should contain the same type IV LTA as S. pneumoniae, providing a more complete understanding of the biosynthesis of these P-Cho-containing TAs in members of the Mitis group of streptococci. Remarkably, we observed besides type IV LTA, an additional polymer belonging to LTA type I in both investigated S. mitis strains. This LTA consists of ß-galactofuranosyl-(1,3)-diacylglycerol as glycolipid anchor and a poly-glycerol-phosphate chain at the O-6 position of the furanosidic galactose. Hence, these bacteria are capable of synthesizing two different LTA polymers, most likely produced by distinct biosynthesis pathways. Our bioinformatics analysis revealed the prevalence of the LTA synthase LtaS, most probably responsible for the second LTA version (type I), among S. mitis and Streptococcus pseudopneumoniae strains.

IL23R on myeloid cells is involved in murine pulmonary granuloma formation.

PURPOSE OF THE STUDY: The involvement of the IL-23/IL23R pathway is well known in the disease pathogenesis of sarcoidosis and other inflammatory diseases. To date, the pathogenic mechanism of IL-23 is most notably described on CD4+ Th17 lymphocytes. However, the function of the IL23R on myeloid cells in sarcoidosis is poorly understood. Thus, the aim of the study is to investigate the role of the IL23R on myeloid cell in pulmonary granuloma formation. Methods: We generated IL23RLysMCre mice lacking the IL23R gene in myeloid cells. The importance of IL23R in myeloid cells for the development of sarcoidosis was studied in a mouse model of inflammatory lung granuloma formation through embolization of PPD from Mycobacterium bovis-coated Sepharose beads into previously PPD-immunized mice. In addition the function of IL23R on myeloid cells was studied in LPS or IFNγ stimulated BMDMs and BMDCs. The mRNA and protein expression levels of relevant cytokines were analyzed by RT-PCR (TaqMan) and ELISA. The composition of immune cells in BALF was quantified by flow cytometry and alteration in granuloma sizes were observed by H&E stained lung sections. Results: Mycobacterium Ag-elicted pulmonary granulomas tend to be smaller in IL23RLysMCre mice and NF-κB dependent Th1 cytokines in the murine lungs are reduced compared to wildtype mice. In line, we observed that IL23R-deficient bone marrow-derived macrophages show a reduced production of Th1 cytokines after LPS stimulation. Conclusion: We here for the first time demonstrate a role for IL23R on myeloid cells in pulmonary inflammation and granuloma formation. Our findings provide essential insights in the pathogenesis of inflammatory lung diseases like sarcoidosis, which might be useful for the development of novel therapeutics targeting distinct immunological pathways like IL-23/IL23R.

Analysis of the Structure and Biosynthesis of the Lipopolysaccharide Core Oligosaccharide of Pseudomonas syringae pv. tomato DC3000.

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, is important for bacterial viability in general and host-pathogen interactions in particular. Negative charges at its core oligosaccharide (core-OS) contribute to membrane integrity through bridging interactions with divalent cations. The molecular structure and synthesis of the core-OS have been resolved in various bacteria including the mammalian pathogen Pseudomonas aeruginosa. A few core-OS structures of plant-associated Pseudomonas strains have been solved to date, but the genetic components of the underlying biosynthesis remained unclear. We conducted a comparative genome analysis of the core-OS gene cluster in Pseudomonas syringae pv. tomato (Pst) DC3000, a widely used model pathogen in plant-microbe interactions, within the P. syringae species complex and to other plant-associated Pseudomonas strains. Our results suggest a genetic and structural conservation of the inner core-OS but variation in outer core-OS composition within the P. syringae species complex. Structural analysis of the core-OS of Pst DC3000 shows an uncommonly high phosphorylation and presence of an O-acetylated sugar. Finally, we combined the results of our genomic survey with available structure information to estimate the core-OS composition of other Pseudomonas species.

Substrate structure-activity relationship reveals a limited lipopolysaccharide chemotype range for intestinal alkaline phosphatase.

Lipopolysaccharide (LPS) from the Gram-negative bacterial outer membrane potently activates the human innate immune system. LPS is recognized by the Toll-like receptor 4/myeloid differentiation factor-2 (TLR4/MD2) complex, leading to the release of pro-inflammatory cytokines. Alkaline phosphatase (AP) is currently being investigated as an anti-inflammatory agent for detoxifying LPS through dephosphorylating lipid A, thus providing a potential treatment for managing both acute (sepsis) and chronic (metabolic endotoxemia) pathologies wherein aberrant TLR4/MD2 activation has been implicated. Endogenous LPS preparations are chemically heterogeneous, and little is known regarding the LPS chemotype substrate range of AP. Here, we investigated the activity of AP on a panel of structurally defined LPS chemotypes isolated from Escherichia coli and demonstrate that calf intestinal AP (cIAP) has only minimal activity against unmodified enteric LPS chemotypes. Pi was only released from a subset of LPS chemotypes harboring spontaneously labile phosphoethanolamine (PEtN) modifications connected through phosphoanhydride bonds. We demonstrate that the spontaneously hydrolyzed O-phosphorylethanolamine is the actual substrate for AP. We found that the 1- and 4'-lipid A phosphate groups critical in TLR4/MD2 signaling become susceptible to hydrolysis only after de-O-acylation of ester linked primary acyl chains on lipid A. Furthermore, PEtN modifications on lipid A specifically enhanced hTLR4 agonist activity of underacylated LPS preparations. Computational binding models are proposed to explain the limitation of AP substrate specificity imposed by the acylation state of lipid A, and the mechanism of PEtN in enhancing hTLR4/MD2 signaling.

Structural analysis and immunostimulatory potency of lipoteichoic acids isolated from three Streptococcus suis serotype 2 strains.

Streptococcus suis serotype 2 is an important porcine and human pathogen. Lipoteichoic acid (LTA) from S. suis has been suggested to contribute to its virulence, and absence of d-alanylation from the S. suis LTA is associated with increased susceptibility to cationic antimicrobial peptides. Here, using high-resolution NMR spectroscopy and MS analyses, we characterized the LTA structures from three S. suis serotype 2 strains differing in virulence, sequence type (ST), and geographical origin. Our analyses revealed that these strains possess-in addition to the typical type I LTA present in other streptococci-a second, mixed-type series of LTA molecules of high complexity. We observed a ST-specific difference in the incorporation of glycosyl residues into these mixed-type LTAs. We found that strains P1/7 (ST1, high virulence) and SC84 (ST7, very high virulence) can attach a 1,2-linked α-d-Glcp residue as branching substituent to an α-d-Glcp that is 1,3-linked to glycerol phosphate moieties and that is not present in strain 89-1591 (ST25, intermediate virulence). In contrast, the latter strain could glycosylate its LTA at the glycerol O-2 position, which was not observed in the other two strains. Using LTA preparations from WT strains and from mutants with an inactivated prolipoprotein diacylglyceryl transferase, resulting in deficient lipoprotein acylation, we show that S. suis LTAs alone do not induce Toll-like receptor 2-dependent pro-inflammatory mediator production from dendritic cells. In summary, our study reveals an unexpected complexity of LTAs present in three S. suis serotype 2 strains differing in genetic background and virulence.

Attachment of phosphorylcholine residues to pneumococcal teichoic acids and modification of substitution patterns by the phosphorylcholine esterase.

The bacterial lung pathogen Streptococcus pneumoniae has a unique nutritional requirement for exogenous choline and attaches phosphorylcholine (P-Cho) residues to the GalpNAc moieties of its teichoic acids (TAs) in its cell wall. Two phosphorylcholine transferases, LicD1 and LicD2, mediate the attachment of P-Cho to the O-6 positions of the two GalpNAc residues present in each repeating unit of pneumococcal TAs (pnTAs), of which only LicD1 has been determined to be essential. At the molecular level, the specificity of the P-Cho attachment to pnTAs by LicD1 and LicD2 remains still elusive. Here, using detailed structural analyses of pnTAs from a LicD2-deficient strain, we confirmed the specificity in the attachment of P-Cho residues to pnTA. LicD1 solely transfers P-Cho to α-d-GalpNAc moieties, whereas LicD2 attaches P-Cho to ß-d-GalpNAc. Further, we investigated the role of the pneumococcal phosphorylcholine esterase (Pce) in the modification of the P-Cho substitution pattern of pnTAs. To clarify the specificity of Pce-mediated P-Cho hydrolysis, we evaluated different concentrations and pH conditions for the treatment of pneumococcal lipoteichoic acid with purified Pce. We show that Pce can hydrolyze both P-Cho residues of the terminal repeat of the pnTA chain and almost all P-Cho residues bound to ß-d-GalpNAc in vitro However, hydrolysis in vivo was restricted to the terminal repeat. In summary, our findings indicate that LicD1 and LicD2 specifically transfer P-Cho to α-d-GalpNAc and ß-d-GalpNAc moieties, respectively, and that Pce removes distinct P-Cho substituents from pnTAs.

Lupus nephritis is linked to disease-activity associated expansions and immunity to a gut commensal.

BACKGROUND/PURPOSE: To search for a transmissible agent involved in lupus pathogenesis, we investigated the faecal microbiota of patients with systemic lupus erythematosus (SLE) for candidate pathobiont(s) and evaluated them for special relationships with host immunity. METHODS: In a cross-sectional discovery cohort, matched blood and faecal samples from 61 female patients with SLE were obtained. Faecal 16 S rRNA analyses were performed, and sera profiled for antibacterial and autoantibody responses, with findings validated in two independent lupus cohorts. RESULTS: Compared with controls, the microbiome in patients with SLE showed decreased species richness diversity, with reductions in taxonomic complexity most pronounced in those with high SLE disease activity index (SLEDAI). Notably, patients with SLE had an overall 5-fold greater representation of Ruminococcus gnavus (RG) of the Lachnospiraceae family, and individual communities also displayed reciprocal contractions of a species with putative protective properties. Gut RG abundance correlated with serum antibodies to only 1/8 RG strains tested. Anti-RG antibodies correlated directly with SLEDAI score and antinative DNA levels, but inversely with C3 and C4. These antibodies were primarily against antigen(s) in an RG strain-restricted pool of cell wall lipoglycans. Novel structural features of these purified lipoglycans were characterised by mass spectrometry and NMR. Highest levels of serum anti-RG strain-restricted antibodies were detected in those with active nephritis (including Class III and IV) in the discovery cohort, with findings validated in two independent cohorts. CONCLUSION: These findings suggest a novel paradigm in which specific strains of a gut commensal may contribute to the immune pathogenesis of lupus nephritis.

Interleukin-1 signaling induced by Streptococcus suis serotype 2 is strain-dependent and contributes to bacterial clearance and inflammation during systemic disease in a mouse model of infection.

Streptococcus suis serotype 2 is an important porcine pathogen and zoonotic agent causing sudden death, septic shock and meningitis, with exacerbated inflammation being a hallmark of the infection. A rapid, effective and balanced innate immune response against S. suis is critical to control bacterial growth without causing excessive inflammation. Even though interleukin (IL)-1 is one of the most potent and earliest pro-inflammatory mediators produced, its role in the S. suis pathogenesis has not been studied. We demonstrated that a classical virulent European sequence type (ST) 1 strain and the highly virulent ST7 strain induce important levels of IL-1 in systemic organs. Moreover, bone marrow-derived dendritic cells and macrophages contribute to its production, with the ST7 strain inducing higher levels. To better understand the underlying mechanisms involved, different cellular pathways were studied. Independently of the strain, IL-1ß production required MyD88 and involved recognition via TLR2 and possibly TLR7 and TLR9. This suggests that the recognized bacterial components are similar and conserved between strains. However, very high levels of the pore-forming toxin suilysin, produced only by the ST7 strain, are required for efficient maturation of pro-IL-1ß via activation of different inflammasomes resulting from pore formation and ion efflux. Using IL-1R-/- mice, we demonstrated that IL-1 signaling plays a beneficial role during S. suis systemic infection by modulating the inflammation required to control and clear bacterial burden, thus promoting host survival. Beyond a certain threshold, however, S. suis-induced inflammation cannot be counterbalanced by this signaling, making it difficult to discriminate its role.

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.

Total Synthesis of Dansylated Park's Nucleotide for High-Throughput MraY Assays.

The membrane protein translocase I (MraY) is a key enzyme in bacterial peptidoglycan biosynthesis. It is therefore frequently discussed as a target for the development of novel antibiotics. The screening of compound libraries for the identification of MraY inhibitors is enabled by an established fluorescence-based MraY assay. However, this assay requires a dansylated derivative of the bacterial biosynthetic intermediate Park's nucleotide as the MraY substrate. Isolation of Park's nucleotide from bacteria and subsequent dansylation only furnishes limited amounts of this substrate, thus hampering the high-throughput screening for MraY inhibitors. Accordingly, the efficient provision of dansylated Park's nucleotide is a major bottleneck in the exploration of this promising drug target. In this work, we present the first total synthesis of dansylated Park's nucleotide, affording an unprecedented amount of the target compound for high-throughput MraY assays.

Accumulation of novel glycolipids and ornithine lipids in Mesorhizobium loti under phosphate deprivation.

Glycolipids are found mainly in photosynthetic organisms (plants, algae, and cyanobacteria), Gram-positive bacteria, and a few other bacterial phyla. They serve as membrane lipids and play a role under phosphate deprivation as surrogates for phospholipids. Mesorhizobium loti accumulates different di- and triglycosyl diacylglycerols, synthesized by the processive glycosyltransferase Pgt-Ml, and two so far unknown glycolipids, which were identified in this study by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy as O-methyl-digalactosyl diacylglycerol (Me-DGD) and glucuronosyl diacylglycerol (GlcAD). Me-DGD is a novel glycolipid, whose synthesis depends on Pgt-Ml activity and the involvement of an unknown methyltransferase, while GlcAD is formed by a novel glycosyltransferase encoded by the open reading frame (ORF) mlr2668, using UDP-glucuronic acid as a sugar donor. Deletion mutants lacking GlcAD are not impaired in growth. Our data suggest that the different glycolipids in Mesorhizobium can mutually replace each other. This may be an adaptation mechanism to enhance the competitiveness in natural environments. A further nonphospholipid in Mesorhizobium was identified as a hydroxylated form of an ornithine lipid with the additional hydroxy group linked to the amide-bound fatty acid, introduced by the hydroxylase OlsD. The presence of this lipid has not been reported for rhizobia yet. The hydroxy group is placed on the C-2 position of the acyl chain as determined by NMR spectroscopy. Furthermore, the isolated ornithine lipids contained up to 80 to 90% d-configured ornithine, a stereoform so far undescribed in bacteria.

NMR-based structural analysis of the complete rough-type lipopolysaccharide isolated from Capnocytophaga canimorsus.

We here describe the NMR analysis of an intact lipopolysaccharide (LPS, endotoxin) in water with 1,2-dihexanoyl-sn-glycero-3-phosphocholine as detergent. When HPLC-purified rough-type LPS of Capnocytophaga canimorsus was prepared, (13)C,(15)N labeling could be avoided. The intact LPS was analyzed by homonuclear ((1)H) and heteronuclear ((1)H,(13)C, and (1)H,(31)P) correlated one- and two-dimensional NMR techniques as well as by mass spectrometry. It consists of a penta-acylated lipid A with an α-linked phosphoethanolamine attached to C-1 of GlcN (I) in the hybrid backbone, lacking the 4'-phosphate. The hydrophilic core oligosaccharide was found to be a complex hexasaccharide with two mannose (Man) and one each of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), Gal, GalN, and l-rhamnose residues. Position 4 of Kdo is substituted by phosphoethanolamine, also present in position 6 of the branched Man(I) residue. This rough-type LPS is exceptional in that all three negative phosphate residues are "masked" by positively charged ethanolamine substituents, leading to an overall zero net charge, which has so far not been observed for any other LPS. In biological assays, the corresponding isolated lipid A was found to be endotoxically almost inactive. By contrast, the intact rough-type LPS described here expressed a 20,000-fold increased endotoxicity, indicating that the core oligosaccharide significantly contributes to the endotoxic potency of the whole rough-type C. canimorsus LPS molecule. Based on these findings, the strict view that lipid A alone represents the toxic center of LPS needs to be reassessed.