In vivo determination of Neisseria meningitidis serogroup A capsular polysaccharide by whole cell high-resolution magic angle spinning NMR spectroscopy

Gudlavalleti, Seshu K.; Szymanski, Christine M.; Jarrell, Harold C.; Stephens, David S.

doi:10.1016/j.carres.2005.11.036

Cited by 34 publications

(23 citation statements)

References 20 publications

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“…Recent improvements in NMR technology permit determination of the capsular PS structure with whole bacteria without the need to purify the PS from clinical isolates (33,34). Also, monoclonal antibodies can be better than polyclonal antibodies in recognizing small serological differences.…”

Section: Discussionmentioning

confidence: 99%

Structure of the Capsular Polysaccharide of Pneumococcal Serotype 11A Reveals a Novel Acetylglycerol That Is the Structural Basis for 11A Subtypes

Zartler

Porambo

Anderson

et al. 2009

Journal of Biological Chemistry

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We have undertaken a structural assessment of Streptococcus pneumoniae 11A polysaccharide as well as two clinical isolates related to 11A. The clinical isolates were labeled 11A␣ and 11A␤. The result of our experiments is a revision to the old structure for S. pneumoniae 11A polysaccharide. The new structure differs from the old structure in both the primary connectivities and acetylation pattern. We also show that 11A contains an acetylglycerol-PO 4 moiety, a substitution that is heretofore unknown in the bacterial polysaccharide literature. The two clinical isolates were also structurally characterized. 11A␣ was determined to be identical to 11A. 11A␤ is a new serotype, which differs from 11A in the absence of the acetylation of the glycerol-PO 4 moiety and a different acetylation pattern of the saccharides. Thus, we propose that the acetylglycerol is the structural basis for 11A␣ and 11A␤ subtypes. The polysaccharide (PS)2 capsule of Streptococcus pneumoniae is recognized as the most important virulence factor of pneumococci. It is expressed by almost all pathogenic pneumococci and has been shown to increase the virulence of a pneumococcal strain by more than a million-fold in an animal model system (1). The capsule shields pneumococci from the host phagocytes, and its shielding capacity can be neutralized when the host produces antibodies to the capsule and to fix complement on pneumococci. Thus, the capsular PS is used as the antigen in all pneumococcal vaccines that are clinically used. Because of their impact on human health, pneumococcal capsules have been extensively investigated both serologically, genetically, and biochemically. Many years of serological studies have identified 91 serologically distinct capsule types (2, 3). Recently, the nucleotide sequences of the capsule gene loci of all 91 different capsule types have been determined (4, 5).With the use of monoclonal antibodies, serologic heterogeneity was noted recently among pneumococcal isolates that were typed as 11A using the quellung reaction (6). To avoid confusion, we have provisionally named the common variant 11A␣ and the less common variant 11A␤. Because 11A PS is a component of the 23-valent pneumococcal PS vaccine (7), it is important to understand the chemical basis of the serological heterogeneity among pneumococci that are typed as serotype 11A. However, there is only limited information on structure of pneumococcal serotype 11A PS, and the structure has not been examined with modern tools. Forty years ago, the structural model for S. pneumoniae serotype 11A PS was proposed to be a linear polymer containing D-glucose, D-galactose, glycerol, phosphate, and O-acetyl groups in the molar ratio of 2:2:1:1:2 based upon analysis of chemically modified PS (8). About 20 years ago, Richards et al. (9,10) proposed the model for 11A PS based on both NMR and chemical degradation studies. They proposed a linear repeating unit structure with four monosaccharides, a pendant glycerol phosphate moiety, and 2 mol of acetate as described in Structure ...

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Section: Discussionmentioning

confidence: 99%

Structure of the Capsular Polysaccharide of Pneumococcal Serotype 11A Reveals a Novel Acetylglycerol That Is the Structural Basis for 11A Subtypes

Zartler

Porambo

Anderson

et al. 2009

Journal of Biological Chemistry

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“…Elution profiles were determined by the phenol-sulphuric method for sugar detection and salt detection by conductivity monitor. The digeneaside fraction was purified by high performance liquid chromatography on a organic acids columns (ICSep ICE-COREGEL-87H3 column, Transgenomic) as shown in Ascencio and Gudlavalleti et al (2006).…”

Section: In Vivo Analyses By Hrmas 1 H Nmr Spectroscopymentioning

confidence: 99%

“…Whereas a number of HRMAS NMR studies have been reported on animal tissue (Moka et al 1997, Bollard et al 2000, Wang et al 2003, higher plant material (Ni and Eads 1993) and bacteria cells (Gudlavalleti et al 2006, Li 2006, few reports have dealt with the use of the technique on algae. By using this in vivo NMR technique Chauton et al (2003) classified several microalgal species and Broberg et al (2000), quantified some metabolites in the red macroalga Gracilariopsis lemaneiformis.…”

Section: Introductionmentioning

confidence: 99%

The use of HRMAS NMR spectroscopy to study the in vivo intra–cellular carbon/nitrogen ratio of Solieria chordalis (Rhodophyta)

et al. 2007

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The red alga Solieria chordalis (J. Agardh) C. Agardh (Rhodophyta) was used as a model to investigate the effects of changes in seawater salinity on the carbon/ nitrogen ratio. Carbohydrates and nitrogenous metabolites are major components of this alga and their metabolisms are intimately linked. Previous publications have provided a solid database for these two primary metabolic pathways from experiments and observations in situ. Storage products (e.g. floridean starch), cell wall polysaccharides (carrageenan) and low molecular weight carbohydrates such as floridoside and digeneaside are major compounds constituting the pool of available carbon. Compounds such as amino acids and peptides, constitute the pool of nitrogen. This study focuses on the intracellular C/N ratio inside the pool of low molecular weight compounds. This C/N ratio can be defined as the balance between carbohydrates and amino acids. High-resolution magic angle spinning nuclear magnetic resonance spectroscopy (HRMAS NMR) provides a powerful approach for in vivo analysis of the pool of intracellular organic compounds. These in vivo results were complimented with quantitative data obtained from high performance liquid chromatography (HPLC). In vivo and in vitro experimental analyses provided a intracellular molecular balance and defined the C/N ratio. In order to study the effect of salt stress on the carbon/nitrogen ratio, S. chordalis was cultured under controlled conditions. Effects of hyposalinity and hypersalinity stresses (low 22 ‰ and high 50 ‰ salinity) were tested. Both HPLC and NMR data, obtained on stressed and unstressed algae, generated insights into variations of carbonated and nitrogenous metabolites, involving changes of the C/N ratios, and demonstrated the adaptive responses of the seaweed.

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“…Cell high-resolution magic angle spinning (HRMAS) NMR spectroscopy can distinguish detailed structures present in bacteria (12,13). Moreover, multidimensional HRMAS NMR is a powerful tool for the in vivo analysis of live bacterial cells (14).…”

Section: Introductionmentioning

confidence: 99%

Live-cell high resolution magic angle spinning magnetic resonance spectroscopy for in vivo analysis of Pseudomonas aeruginosa metabolomics

et al. 2013

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Abstract. Pseudomonas aeruginosa (PA) is a pathogenic gram-negative bacterium that is widespread in nature, inhabiting soil, water, plants and animals. PA is a prevalent cause of deleterious human infections, particularly in patients whose host defense mechanisms have been compromised. Metabolomics is an important tool used to study host-pathogen interactions and to identify novel therapeutic targets and corresponding compounds. The aim of the present study was to report the metabolic profile of live PA bacteria using in vivo high-resolution magic angle spinning (HRMAS) nuclear magnetic resonance spectroscopy (NMR), in combination with 1-and 2-dimensional HRMAS NMR. This methodology provides a new and powerful technique to rapidly interrogate the metabolome of intact bacterial cells and has several advantages over traditional techniques that identify metabolome components from disrupted cells. Furthermore, application of multidimensional HRMAS NMR, in combination with the novel technique total through-Bond correlation Spectroscopy (TOBSY), is a promising approach that may be used to obtain in vivo metabolomics information from intact live bacterial cells and can mediate such analyses in a short period of time. Moreover, HRMAS 1 H NMR enables the investigation of the associations between metabolites and cell processes. In the present study, we detected and quantified several informative metabolic molecules in live PA cells, including N-acetyl, betaine, citrulline, alanine and glycine, which are important in peptidoglycan synthesis. The results provided a complete metabolic profile of PA for future studies of PA clinical isolates and mutants. In addition, this in vivo NMR biomedical approach might have clinical utility and should prove useful in gene function validation, the study of pathogenetic mechanisms, the classification of microbial strains into functional/clinical groups, the testing of anti-bacterial agents and the determination of metabolic profiles of bacterial mutants. IntroductionPseudomonas aeruginosa (PA) is a ubiquitous Gram-negative bacterium that inhabits a wide array of natural environments. This bacterium is of significant clinical interest as it is a multi-antibiotic-resistant human pathogen associated with hospital-acquired infections (1,2) and a major cause of morbidity and mortality in cystic fibrosis (CF) patients. It commonly colonizes the lower respiratory and gastrointestinal tracts and the mucosa and skin of hospital patients. The establishment of chronic PA respiratory infections requires a complex adaptive process that mediates essential physiological changes that allow bacterial cells to survive and persist in the host environment. Bacteria secrete small molecules that act as specific signals to positively regulate specialized processes (3), including the production of virulence factors that mediate pathogenic infection, host colonization and the promotion of interspecies microbial interactions (4).Bacterial cell walls are complex, consisting of integrated macromolecules including c...

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In vivo determination of Neisseria meningitidis serogroup A capsular polysaccharide by whole cell high-resolution magic angle spinning NMR spectroscopy

Cited by 34 publications

References 20 publications

Structure of the Capsular Polysaccharide of Pneumococcal Serotype 11A Reveals a Novel Acetylglycerol That Is the Structural Basis for 11A Subtypes

Structure of the Capsular Polysaccharide of Pneumococcal Serotype 11A Reveals a Novel Acetylglycerol That Is the Structural Basis for 11A Subtypes

The use of HRMAS NMR spectroscopy to study the in vivo intra–cellular carbon/nitrogen ratio of Solieria chordalis (Rhodophyta)

Live-cell high resolution magic angle spinning magnetic resonance spectroscopy for in vivo analysis of Pseudomonas aeruginosa metabolomics

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