Evidence for a LOS and a capsular polysaccharide in Capnocytophaga canimorsus

Capnocytophaga canimorsus is a dog's and cat's oral commensal which can cause fatal human infections upon bites or scratches. Infections mainly start with flu-like symptoms but can rapidly evolve in fatal septicaemia with a mortality as high as 40%. Here we present the discovery of a polysaccharide capsule (CPS) at the surface of C. canimorsus 5 (Cc5), a strain isolated from a fulminant septicaemia. We provide genetic and chemical data showing that this capsule is related to the lipooligosaccharide (LOS) and probably composed of the same polysaccharide units. A CPS was also found in nine out of nine other strains of C. canimorsus. In addition, the genomes of three of these strains, sequenced previously, contain genes similar to those encoding CPS biosynthesis in Cc5. Thus, the presence of a CPS is likely to be a common property of C. canimorsus. The CPS and not the LOS confers protection against the bactericidal effect of human serum and phagocytosis by macrophages. An antiserum raised against the capsule increased the killing of C. canimorsus by human serum thus showing that anti-capsule antibodies have a protective role. These findings provide a new major element in the understanding of the pathogenesis of C. canimorsus

Early detection of psychosis: helpful or stigmatizing experience? A qualitative study

Aim: Despite the large scientific debate concerning potential stigmatizing effects of identifying an individual as being in an at‐risk mental state (ARMS) for psychosis, studies investigating this topic from the subjective perspective of patients are rare. This study assesses whether ARMS individuals experience stigmatization and to what extent being informed about the ARMS is experienced as helpful or harmful. Methods Eleven ARMS individuals, currently participating in the follow‐up assessments of the prospective Basel Früherkennung von Psychosen (FePsy; English: Early Detection of Psychosis) study, were interviewed in detail using a semistructured qualitative interview developed for this purpose. Data were analysed using Interpretative Phenomenological Analysis. Results Most individuals experiencing first symptoms reported sensing that there was 'something wrong with them' and felt in need of help. They were relieved that a specific term was assigned to their symptoms. The support received from the early detection centre was generally experienced as helpful. Many patients reported stigmatization and discrimination that appeared to be the result of altered behaviour and social withdrawal due to the prepsychotic symptoms they experienced prior to contact with the early detection clinic. Conclusions The results suggest that early detection services help individuals cope with symptoms and potential stigmatization rather than enhancing or causing the latter. More emphasis should be put on the subjective experiences of those concerned when debating the advantages and disadvantages of early detection with regard to stigma. There was no evidence for increased perceived stigma and discrimination as a result of receiving information about the ARMS

Extrapolation for Time-Series and Cross-Sectional Data

Extrapolation methods are reliable, objective, inexpensive, quick, and easily automated. As a result, they are widely used, especially for inventory and production forecasts, for operational planning for up to two years ahead, and for long-term forecasts in some situations, such as population forecasting. This paper provides principles for selecting and preparing data, making seasonal adjustments, extrapolating, assessing uncertainty, and identifying when to use extrapolation. The principles are based on received wisdom (i.e., experts’ commonly held opinions) and on empirical studies. Some of the more important principles are:• In selecting and preparing data, use all relevant data and adjust the data for important events that occurred in the past.• Make seasonal adjustments only when seasonal effects are expected and only if there is good evidence by which to measure them.• In extrapolating, use simple functional forms. Weight the most recent data heavily if there are small measurement errors, stable series, and short forecast horizons. Domain knowledge and forecasting expertise can help to select effective extrapolation procedures. When there is uncertainty, be conservative in forecasting trends. Update extrapolation models as new data are received.• To assess uncertainty, make empirical estimates to establish prediction intervals.• Use pure extrapolation when many forecasts are required, little is known about the situation, the situation is stable, and expert forecasts might be biased

The Lipopolysaccharide from Capnocytophaga canimorsus Reveals an Unexpected Role of the Core-Oligosaccharide in MD-2 Binding

Capnocytophaga canimorsus is a usual member of dog's mouths flora that causes rare but dramatic human infections after dog bites. We determined the structure of C. canimorsus lipid A. The main features are that it is penta-acylated and composed of a “hybrid backbone” lacking the 4′ phosphate and having a 1 phosphoethanolamine (P-Etn) at 2-amino-2-deoxy-d-glucose (GlcN). C. canimorsus LPS was 100 fold less endotoxic than Escherichia coli LPS. Surprisingly, C. canimorsus lipid A was 20,000 fold less endotoxic than the C. canimorsus lipid A-core. This represents the first example in which the core-oligosaccharide dramatically increases endotoxicity of a low endotoxic lipid A. The binding to human myeloid differentiation factor 2 (MD-2) was dramatically increased upon presence of the LPS core on the lipid A, explaining the difference in endotoxicity. Interaction of MD-2, cluster of differentiation antigen 14 (CD14) or LPS-binding protein (LBP) with the negative charge in the 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) of the core might be needed to form the MD-2 – lipid A complex in case the 4′ phosphate is not present

The surface structures of "Capnocytophaga canimorsus" and their role in pathogenesis

Since 1976 there have been numerous case reports about severe sepsis or meningitis in humans after dog bites or scratches. The bacterium causing these dramatic infections was identified as Capnocytophaga canimorsus. C. canimorsus belong to the family of Flavobacteriaceae in the phylum Bacteroidetes and are usual members of dog’s mouth flora. Human infections are rare and occur with an approximate frequency of one case per million inhabitants and year. The surface polysaccharides of commensal as well as pathogenic bacteria have to fulfil a multitude of functions to ensure viability. Besides phospholipids the outer membrane of Gram-negative bacteria consists of a unique carbohydrate component, the lipopolysaccharide (LPS). The LPS of gram-negative bacteria consists of three regions: the lipid A, the core-oligosaccharide, and the O-antigen. This work describes the structure of C. canimorsus lipid A, core-oligosaccharide and O-antigens. The main features of the lipid A are that it is penta-acylated and composed of a ”hybrid backbone“ lacking the 4’ phosphate and having a 1-P-Etn at GlcN. C. canimorsus LPS was 100 fold less endotoxic than Escherichia coli LPS. Surprisingly, C. canimorsus lipid A was 20,000 fold less endotoxic than the C. canimorsus lipid A-core. This represents the first example in which the core-oligosaccharide dramatically increases endotoxicity of a low endotoxic lipid A. The binding to human MD-2 was dramatically increased upon presence of the LPS core on the lipid A, explaining the difference in endotoxicity. Interaction of MD-2 or LBP/CD14 with the negative charge in the Kdo of the core might be needed to form the MD-2 – lipid A complex in case the 4’ phosphate is not present. Overall the properties of the lipid A-core may explain how this bacterium first escapes recognition by receptors of the innate immune system, but nevertheless is able to provoke a shock at the septic stage. We further show that the C. canimorsus genome encodes in a single operon a lipid A 1 phosphatase (LpxE) and a lipid A 1 P-Etn transferase (EptA). This suggests that LPS is modified after its synthesis by removal of the 1 phosphate and subsequent addition of a P-Etn group. In agreement with this prediction, deletion of lpxE or eptA led to increased endotoxicity and decreased resistance to cationic antimicrobial peptides (CAMP), where deletion of lpxE had a more severe effect. The endotoxicity and CAMP resistance of a double deletion mutant of lpxE-eptA was similar to that of a single lpxE mutant. The structure of the complete LPS from C. canimorsus 5 (Cc5) was determined by chemical analysis, GLC-MS, ESI FT-ICR MS and NMR spectroscopy. Two different O-antigens (LPS I and LPS II) were found to be co-expressed. LPS I consists of repeating units of N-Acetylfucosamine (FucNAc), glucuronic acid (GlcA), N-Acetylquinovosamine (QuiNAc) and N-galacturonoyl-2-aminoglycerol (GalANgro) while LPS II O-antigen consists of five repeating units of N-Acetylglucosamine (GlcNAc) and L-Rhamnose (L-Rha). Several transposon mutants sensitive to complement killing isolated by a large screen turned out to be also sensitive to killing by Polymyxin B. All the mutations mapped in a 28-kb locus consisting of 29 genes involved in the biosynthesis and assembly of the sugars identified in LPS I and LPS II. All serum- and Polymyxin-sensitive mutants lacked LPS I but also a high molecular weight polysaccharide reacting with a specific anti LPS I antiserum. We inferred that this polysaccharide was a type 1 or 4 capsule consisting of the LPS I repeating units. The K-antigen, formed by LPS I and the related capsule, but not LPS II, were found to be assembled by a wzx/wzy dependent process. Deletion of wzz lead to deregulation of the length of LPS I, to the loss of the LPS I dependent capsule and to an altered surface as detected by TEM. Summarizing, we show that the C. canimorsus 5 K-antigen is responsible for the complement and Polymyxin B resistance

Chemical synthesis of Burkholderia Lipid A modified with glycosyl phosphodiester-linked 4-amino-4-deoxy-β-L-arabinose and its immunomodulatory potential

Modification of the Lipid A phosphates by positively charged appendages is a part of the survival strategy of numerous opportunistic Gram-negative bacteria. The phosphate groups of the cystic fibrosis adapted Burkholderia Lipid A are abundantly esterified by 4-amino-4-deoxy-β-L-arabinose (β-L-Ara4N), which imposes resistance to antibiotic treatment and contributes to bacterial virulence. To establish structural features accounting for the unique pro-inflammatory activity of Burkholderia LPS we have synthesised Lipid A substituted by β-L-Ara4N at the anomeric phosphate and its Ara4N-free counterpart. The double glycosyl phosphodiester was assembled by triazolyl-tris-(pyrrolidinyl)phosphonium-assisted coupling of the β-L-Ara4N H-phosphonate to α-lactol of β(1→6) diglucosamine, pentaacylated with (R)-(3)-acyloxyacyl- and Alloc-protected (R)-(3)-hydroxyacyl residues. The intermediate 1,1'-glycosyl-H-phosphonate diester was oxidised in anhydrous conditions to provide, after total deprotection, β-L-Ara4N-substituted Burkholderia Lipid A. The β-L-Ara4N modification significantly enhanced the pro-inflammatory innate immune signaling of otherwise non-endotoxic Burkholderia Lipid A

Modification of the 1-phosphate group during biosynthesis of Capnocytophaga canimorsus lipid A

Capnocytophaga canimorsus, a commensal bacterium of dog's mouth flora causing severe infections in humans after dog bites or scratches, has a lipopolysaccharide (LPS, endotoxin) with low-inflammatory lipid A. In particular it contains a phosphoethanolamine (P-Etn) instead of a free phosphate group at the C-1 position of the lipid A backbone, usually present in highly toxic enterobacterial Gram-negative lipid A. Here we show that the C. canimorsus genome comprises a single operon encoding a lipid A 1-phosphatase (LpxE) and a lipid A 1 P-Etn transferase (EptA). This suggests that lipid A is modified during biosynthesis after completing acylation of the backbone by removal of the 1-phosphate and subsequent addition of a P-Etn group. As endotoxicity of lipid A is known to depend largely on the degree of unsubstituted or unmodified phosphate residues, deletion of lpxE or eptA led to mutants lacking the P-Etn group, with consequently increased endotoxicity and decreased resistance to cationic antimicrobial peptides (CAMP). Consistent with the proposed sequential biosynthetic mechanism, the endotoxicity and CAMP resistance of a double deletion mutant of lpxE-eptA was similar to that of a single lpxE mutant. Finally, the proposed enzymatic activities of LpxE and EptA based on sequence similarity could be successfully validated by MS-based analysis of lipid A isolated from corresponding deletion mutant strains

Conformationally constrained Lipid A mimetics for exploration of structural basis of TLR4/MD-2 activation by lipopolysaccharide

Recognition of the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, by the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD-2) complex is essential for the control of bacterial infection. A pro-inflammatory signaling cascade is initiated upon binding of membrane-associated portion of LPS, a glycophospholipid Lipid A, by a co-receptor protein MD-2, which results in a protective host innate immune response. However, activation of TLR4 signaling by LPS may lead to the dysregulated immune response resulting in a variety of inflammatory conditions including sepsis syndrome. Understanding of structural requirements for Lipid A endotoxicity would ensure the development of effective anti-inflammatory medications. Herein we report on design, synthesis and biological activities of a series of conformationally confined Lipid A mimetics based on β,α-trehalose-type scaffold. Replacement of the flexible three-bond β(1→6) linkage in diglucosamine backbone of Lipid A by a two-bond β,α(1↔1) glycosidic linkage afforded novel potent TLR4 antagonists. Synthetic tetraacylated bisphosphorylated Lipid A mimetics based on a β-GlcN(1↔1)α-GlcN scaffold selectively block the LPS binding site on both human and murine MD-2 and completely abolish lipopolysaccharide-induced pro-inflammatory signaling, thereby serving as antisepsis drug candidates. In contrast to their natural counterpart lipid IVa, conformationally constrained Lipid A mimetics do not activate mouse TLR4. The structural basis for high antagonistic activity of novel Lipid A mimetics was confirmed by molecular dynamics simulation. Our findings suggest that besides the chemical structure, also the three-dimensional arrangement of the diglucosamine backbone of MD-2-bound Lipid A determines endotoxic effects on TLR4