Polysaccharide Region Research Articles

Structural elucidation of the O-antigen polysaccharide from shigatoxin-producing E. coli O179 using genetic information, NMR spectroscopy and the CASPER program.

The serological properties of the O-antigen polysaccharide region of the lipopolysaccharides are used to differentiate E. coli strains into serogroups. In this study, we report the structure elucidation of the O-specific chain of E. coli O179 using NMR data, the program CASPER and analysis of biosynthetic information available in the E. coli O-antigen Database (ECODAB). The presence of genes that encode enzymes involved in the biosynthesis of the GDP-Man and UDP-GlcA within the O-antigen gene cluster of the bacteria indicates that the corresponding residues could be present in the polysaccharide. Furthermore, the occurrence of four genes that encode for glycosyltransferases indicates that the polysaccharide is composed of pentasaccharide repeating units; a bioinformatics approach based on predictive glycosyltransferase functions present in ECODAB revealed that the β-d-Manp-(1→4)-β-d-Manp-(1→3)-d-GlcpNAc structural element could be present in the O-specific chain. NMR spectroscopy data obtained from homonuclear and heteronuclear 2D NMR spectra (1H,1H-TOCSY, 1H,13C-HSQC, 1H,13C-H2BC and 1H,13C-HMBC) were analyzed using the CASPER program, revealing the following arrangement of monosaccharide residues as the most probable structure: →4)-α-d-GlcpA-(1→3)-[β-d-Glcp-(1→2)]β-d-Manp-(1→4)-β-d-Manp-(1→3)-β-d-GlcpNAc-(1→, which was further confirmed using 2D homonuclear 1H,1H-COSY and 1H,1H-NOESY spectra. The functions of the α-gluconosyltransferase and the β-glucosyltransferase were predicted using structural alignment of AlphaFold-predicted 3D structures. This O-antigen polysaccharide shares structural similarities with those of E. coli O6 and O188, S. boydii type 16, and the capsular polysaccharide of E. coli K43, explaining the serological cross-reactivities observed with strains belonging these O- and K-antigen groups.

Elucidation of the structural changes of hemicellulose and cellulose in sunflower seed during roasting.

This study evaluated the structural changes in hemicellulose and cellulose from sunflower seeds before and after roasting at 160°C, 190°C, and 220°C. Sugar composition, molecular weight, Fourier transform infrared spectrometry, thermogravimetric, and NMR analyses were utilized to determine the structural properties of these polysaccharides and detect the volatile compounds. The results showed that roasting destroyed the microstructure of these hemicelluloses and cellulose. Glucose and arabinose of hemicellulose were more easily degraded than other sugars during roasting. The galacturonic acid content increased from 7.8% to 46.66% after roasting. The hemicellulose obtained at 220°C had a backbone of D-xylose residues with a β-(1→4)-linkage. The molecular weight of cellulosic polysaccharides decreased with the increase of roasting temperature. The crystallinity increased from 28.92% to 31.86% revealing that mainly the amorphous regions of cellulosic polysaccharides were destroyed by roasting. After roasting, the volatile compounds of these polysaccharides were rich in furfural, which was produced by caramelization and the Maillard reaction, contributing to the characteristic aroma of roasted sunflower seeds. This study provides some information on the relationship between structural changes of polysaccharides and the formation of flavor during roasting sunflower seeds.

Structural analyses of apricot pectin polysaccharides

Apricot pectin polysaccharides' fine structure was performed using HPSEC, HPAEC-PAD, GC–MS, NMR and FTIR spectroscopies. Purified pectin fraction (F1AP) was composed of D-galacturonic acid, L-rhamnose, D-arabinose and D-galactose, Mw ∼ 1588 kDa. F1AP was eluted by water and with 0.2 M NaCl from DEAE Sepharose fraction resulting in two distinct fractions, F1AP1 and F1AP6, with different structures, molecular weights, and conformations, providing insights into their structural diversity. F1AP1 neutral properties were related to its association with protein. F1AP1 had a backbone of (1 → 4)-linked-D-galacturonic acid and (1 → 2)-linked-L-rhamnopyranosyl residues branched with arabinogalactan including multiple glycosidic linkages of T-α-Araf, 3-α-Araf, 5-α-Araf, T-α-Arap, 2-α-Arap, t-Galp, 2-Galp, 3-Galp, 4-Galp, 6-Galp, 2,4-Galp, 3,4-Galp, 3,6-Galp and 4,6-Galp side chains, having methyl and acetylated groups, and a high molecular weight (1945 kDa). The Mark-Houwink exponent was 0.276, indicating a compact spherical conformation. While the other F1AP6 fraction consists predominately of less methylated HG regions of pectin polysaccharides. The molar mass of this fraction was 117.5 kDa, which adopted a stiffer and random coil conformation. This knowledge allows us to evaluate how the balance of chemical structure and physical properties of the two pectin domains may manifest itself in the isolated structure of apricot pectin and its applications.

Global biochemical profiling of fast-growing Antarctic bacteria isolated from meltwater ponds by high-throughput FTIR spectroscopy.

Fourier transform infrared (FTIR) spectroscopy is a biophysical technique used for non-destructive biochemical profiling of biological samples. It can provide comprehensive information about the total cellular biochemical profile of microbial cells. In this study, FTIR spectroscopy was used to perform biochemical characterization of twenty-nine bacterial strains isolated from the Antarctic meltwater ponds. The bacteria were grown on two forms of brain heart infusion (BHI) medium: agar at six different temperatures (4, 10, 18, 25, 30, and 37°C) and on broth at 18°C. Multivariate data analysis approaches such as principal component analysis (PCA) and correlation analysis were used to study the difference in biochemical profiles induced by the cultivation conditions. The observed results indicated a strong correlation between FTIR spectra and the phylogenetic relationships among the studied bacteria. The most accurate taxonomy-aligned clustering was achieved with bacteria cultivated on agar. Cultivation on two forms of BHI medium provided biochemically different bacterial biomass. The impact of temperature on the total cellular biochemical profile of the studied bacteria was species-specific, however, similarly for all bacteria, lipid spectral region was the least affected while polysaccharide region was the most affected by different temperatures. The biggest temperature-triggered changes of the cell chemistry were detected for bacteria with a wide temperature tolerance such Pseudomonas lundensis strains and Acinetobacter lwoffii BIM B-1558.

Mechanistic insight into the aggregation ability of anammox microorganisms: Roles of polarity, composition and molecular structure of extracellular polymeric substances

The chemical characteristics of extracellular polymeric substances (EPS) of anammox bacteria (AnAOB) play a crucial role in the rapid enrichment of AnAOB and the stable operation of wastewater anammox processes. To clarify the influential mechanisms of sludge EPS on AnAOB aggregation, multiple parameters, including the polarity distribution, composition, and molecular structure of EPS, were selected, and their quantitative relationship with AnAOB aggregation was analyzed. Compared to typical anaerobic sludge (anaerobic floc and granular sludge), the anammox sludge EPS exhibited higher levels of tryptophan-like substances (44.82–56.52 % vs. 2.57–39.81 %), polysaccharides (40.02–53.49 mg/g VSS vs. 30.22–41.69 mg/g VSS), and protein structural units including α-helices (20.70–23.98 % vs. 16.48–19.32 %), β-sheets (37.43–42.98 % vs. 25.78–36.72 %), and protonated nitrogen (Npr) (0.065–0.122 vs. 0.017–0.061). In contrast, it had lower contents of β-turns (20.95–27.39 % vs. 28.17–39.04 %). These biopolymers were found to originate from different genera of AnAOB. Specifically, the α-helix-rich proteins were mainly derived from Candidatus Kuenenia, whereas the extracellular proteins related to tryptophan and Npr were closely associated with Candidatus Brocadia. Critically, these EPS components could drive anammox aggregation through interactions. Substantial amounts of tryptophan-like substances facilitated the formation of β-sheet structures and the exposure of internal hydrophobic clusters, which benefited the anammox aggregation. Meanwhile, extracellular proteins with high Npr content played a pivotal role in the formation of mixed protein-polysaccharide gel networks with the electronegative regions of polysaccharides, which could be regarded as the key component in the maintenance of anammox sludge stability. These findings provide a comprehensive understanding of the multifaceted roles of EPS in driving anammox aggregation and offer valuable insights into the development of EPS regulation strategies aimed at optimizing the anammox process.

Prevalence of multidrug-resistant hypervirulent Klebsiella pneumoniae without defined hypervirulent biomarkers in Anhui, China: a new dimension of hypervirulence.

Klebsiella pneumoniae is an opportunistic pathogen that mainly causes nosocomial infections and hospital-associated pneumonia in elderly and immunocompromised people. However, multidrug-resistant hypervirulent K. pneumoniae (MDR-hvKp) has emerged recently as a serious threat to global health that can infect both immunocompromised and healthy individuals. It is scientifically established that plasmid-mediated regulator of mucoid phenotype genes (rmpA and rmpA2) and other virulence factors (aerobactin and salmochelin) are mainly responsible for this phenotype. In this study, we collected 23 MDR-hvKp isolates and performed molecular typing, whole genome sequencing, comparative genomic analysis, and phenotypic experiments, including the Galleria mellonella infection model, to reveal its genetic and phenotypic features. Meanwhile, we discovered two MDR-hvKp isolates (22122315 and 22091569) that showed a wide range of hypervirulence and hypermucoviscosity without rmpA and rmpA2 and any virulence factors. In phenotypic experiments, isolate 22122315 showed the highest hypervirulence (infection model) with significant mucoviscosity, and conversely, isolate 22091569 exhibited the highest mucoviscosity (string test) with higher virulence compared to control. These two isolates carried carbapenemase (blaKPC - 2), β-lactamase (blaOXA - 1, blaTEM - 1B), extended-spectrum β-lactamase (ESBL) genes (blaCTX - M - 15, blaSHV - 106), outer membrane protein-coding genes (ompA), fimbriae encoding genes (ecpABCDER), and enterobactin coding genes (entAB, fepC). In addition, single nucleotide polymorphism analysis indicated that both isolates, 22122315 and 22091569, were found to have novel mutations in loci FEBNDAKP_03184 (c. 2084A > C, p. Asn695Thr), and EOFMAFIB_02276 (c. 1930C > A, p. Pro644Thr), respectively. Finally, NCBI blast analysis suggested these mutations are located in the wzc of the capsule polysaccharide (cps) region and are responsible for putative tyrosine kinase. This study would be a strong reference for enhancing the current understanding of identifying the MDR-hvKp isolates that lacked both mucoid regulators and virulence factors.

Polysaccharide-Targeting Lipid Nanoparticles to Kill Gram-Negative Bacteria.

The rapid increase and spread of Gram-negative bacteria resistant to many or all existing treatments threaten a return to the preantibiotic era. The presence of bacterial polysaccharides that impede the penetration of many antimicrobials and protect them from the innate immune system contributes to resistance and pathogenicity. No currently approved antibiotics target the polysaccharide regions of microbes. Here, describe monolaurin-based niosomes, the first lipid nanoparticles that can eliminate bacterial polysaccharides from hypervirulent Klebsiella pneumoniae, are described. Their combination with polymyxin B shows no cytotoxicity in vitro and is highly effective in combating K. pneumoniae infection in vivo. Comprehensive mechanistic studies have revealed that antimicrobial activity proceeds via a multimodal mechanism. Initially, lipid nanoparticles disrupt polysaccharides, then outer and inner membranes are destabilized and destroyed by polymyxin B, resulting in synergistic cell lysis. This novel lipidic nanoparticle system shows tremendous promise as a highly effective antimicrobial treatment targeting multidrug-resistant Gram-negative pathogens.

Structural changes of cellulosic polysaccharides in sesame kernels during roasting

SummaryThis study investigated the structural changes of sesame kernel cellulosic polysaccharides during roasting and identified the volatile products produced from the degradation of cellulosic polysaccharides. The results showed that rhamnose and galacturonic acid of cellulosic polysaccharides were more susceptible to non‐enzymatic browning reaction than glucose. The degradation of cellulosic polysaccharides in the roasting temperature range of 180–220°C resulted in decreasing in their molecular weight from 163 270 to 125 477 Da. The crystallinity increased from 16.4% to 32.5% revealing that mainly the amorphous regions of cellulosic polysaccharides were degraded during roasting. In addition, the volatiles from degradation of the amorphous region were rich in aldehydes (10.17–33.33%) and furans (12.84–41.55%). This study provides a new perspective for improving the quality of sesame kernel products in the food industry.

Prediction of Pasteurella multocida serotypes based on whole genomic sequences

The serotypes of Pasteurella multocida were predicted based on whole genomic sequences (WGS) with specific genes of the capsular and liposaccharide (LPS) outer core polysaccharide regions as targets. A total of 56 strains were whole genomic sequenced and in addition all assembled genomes from NCBI were included for comparison. BIGSdb (Bacterial Isolate Genome Sequence Database) was installed on a Linux server and targets for capsular types A, B, D, E and F were defined as gene sequences of hyaD, bcbD, dcbF, ecbJ and fcbD, respectively and targets for LPS groups 1, 2, 3, 4, 5, 6, 7 and 8 were defined as gene sequences of pcgB, nctA, gatF, latB, rmlA, nctB, ppgB and natG, respectively. The serotypes of P. multocida were predicted from WGS by designating the capsular type and LPS group as well as subtype alleles to isolates. Comparisons between WGS predictions of capsular types and classical phenotypic typing showed correspondence in 87 % of cases whereas comparisons of WGS predictions of LPS groups to phenotypic typing corresponded for 82 % of the strains. In total 93 % and 94 % of the strains available with WGS could be capsular and LPS group typed, respectively. The server is free to access from https://ivsmlst.sund.ku.dk.

In-vitro digestion and fermentation of cranberry extracts rich in cell wall oligo/polysaccharides

• Cranberry extracts rich in cell wall oligo/polysaccharides and polyphenols were produced and characterised. • Pectic polysaccharide regions of cranberry extract resisted the gastrointestinal digestion, while hemicellulosic polysaccharides were hydrolyzed. • Digestion increases bioavailability of polyphenolic compounds. • Enzymatically hydrolyzed cranberry extract increased the population of Bifidobacterium . • Enterobacteriaceae decreased upon fermentation of cranberry extracts. • Acetate, propionate, and butyrate were produced upon the fermentation of cranberry extracts. Cranberry extracts, generated by microwave-assisted extraction and enzymatic-treatment, were subjected to in-vitro- gastrointestinal digestion and colonic fermentation using human-fecal-inoculum. 54.3% of neutral sugars were found in jejunum dialysate, whereas 51.2% of uronic acids were recovered in effluents. Pectic polysaccharide regions of cranberry extract resisted the gastrointestinal digestion, while hemicellulosic polysaccharides were hydrolyzed. Total polyphenolic content in all digesta was 2.3 times higher as compared to feed meal. Cranberry products (pomace-extract-hydrolysate) showed different fermentation behaviors and were compared to inulin-type-oligofructose. All cranberry products improved the growth of Clostridia cluster-XIVa ( Δ logbacteria = 1.3–2.5). Cranberry hydrolysate and oligofructose increased the population of Bifidobacterium spp ( Δ log = 0.5–0.6). Contrary to pomace and oligofructose, cranberry extract and its hydrolysate did not enhance the growth of Enterobacteriaceae . Short-chain-fatty acid analysis revealed the production of large amounts of acetate, propionate, and butyrate with total concentrations of 145.5–161.7 mmol/L. This study lays the ground for the development of prebiotic-cranberry extracts as value-added-functional ingredients.

Protein FT-IR amide bands are beneficial to bacterial typing

Bacterial FT-IR signals are extremely specific and highly reproducible, making FT-IR an efficient tool for bacterial typing at the subspecies level. The polysaccharide and nucleic acid FT-IR regions (1200–900 cm−1) are recommended as a precise and reproducible pattern for bacterial typing. However, proteins are the major macromolecules present in bacteria, and the FT-IR spectral region of proteins (1800–1300 cm−1) is conceivably an important factor in bacterial typing. In this study, we investigated the influence of water on bacterial protein amide bands by comparing spectra obtained with and without FT-IR system dehydration. Eight Escherichia coli, ten Klebsiella pneumoniae, and eleven Staphylococcus aureus strains were typed by FT-IR under different conditions in a blinded experimental setup. Hierarchical clustering analysis (HCA) showed that, when protein signals were included (1800–900 cm−1), the typing accuracies for select E. coli, K. pn and S. aureus strains without system dehydration were 50%, 30% and 18.2%, respectively. However, the accuracies greatly improved to 100%, 90% and 90.9% when the FT-IR system was dehydrated. These results indicate that the FT-IR signals of protein amide bands are beneficial for bacterial typing.

Influence of the Sample Preparation Method in Discriminating Candida spp. Using ATR-FTIR Spectroscopy.

Several studies have investigated the capacity of ATR-FTIR spectroscopy for fungal species discrimination. However, preparation methods vary among studies. This study aims to ascertain the effect of sample preparation on the discriminatory capacity of ATR-FTIR spectroscopy. Candida species were streaked to obtain colonies and spectra were collected from each preparation type, which included: (a) untreated colonies being directly transferred to the ATR crystal, (b) following washing and (c) following 24-h fixation in formalin. Spectra were pre-processed and principal component analysis (PCA) and K-means cluster analysis (KMC) were performed. Results showed that there was a clear discrimination between preparation types. Groups of spectra from untreated and washed isolates clustered separately due to intense protein, DNA and polysaccharide bands, whilst fixed spectra clustered separately due to intense polysaccharide bands. This signified that sample preparation had influenced the chemical composition of samples. Nevertheless, across preparation types, significant species discrimination was observed, and the polysaccharide (1200–900 cm−1) region was a common critical marker for species discrimination. However, different discriminatory marker bands were observed across preparation methods. Thus, sample preparation appears to influence the chemical composition of Candida samples; however, does not seem to significantly impact the species discrimination potential for ATR-FTIR spectroscopy.

The Structure Features and Improving Effects of Polysaccharide from Astragalus membranaceus on Antibiotic-Associated Diarrhea.

Astragalus membranaceus (Astragalus) is often used as a medical and food resource in China. The present study was designed to investigate the features and effects of polysaccharide from Astragalus membranaceus (WAP) on rats with antibiotic-associated diarrhea (AAD). WAP was mainly composed of glucose, galactose, arabinose and glacturonic acid, with glucan, arabinogalactan and RG-I regions, and it showed loosely irregular sheet conformation. WAP decreased the inflammatory cell infiltration of colon in AAD rats, increased propionate and butyrate production, improved metabolic levels, adjusted the diversity and composition of gut microbiota, increased the relative abundance of Pseudomonas, and decreased the relative abundance of Allobaculum and Coprococcus. In conclusion, WAP contained different types of polysaccharide regions and sheet three-dimensional conformation, while it ameliorated AAD by recovering the colon structure, adjusting the gut microbiota, and improving the SCFAs levels. The results can provide some data basis for natural products to alleviate the side effects related to antibiotics.

The strategy for correcting interference from water in Fourier transform infrared spectrum based bacterial typing

Accurate and effective identification and typing of microorganisms is important in epidemiological surveillance. Fourier transform infrared (FTIR) spectroscopy is a promising bacterial typing method based on extracting the infrared spectrum signal-related biochemical features of intact microbiological cells. Unfortunately, the FTIR signals of bacteria are disturbed by many factors, especially the unavoidable absorbance of H2O, and many studies have focused only on the internal biochemical information. In this study, the interference from water was analyzed and verified by experimental data. The infrared absorbance of H2O overlapped with the protein (1200-1800 cm−1) and lipid regions (2800-3700 cm−1), but had little impact on the polysaccharide and nucleic acid region (900-1200 cm−1). The elimination of the protein and lipid region markedly decreased the interference of H2O and increased the typing accuracy. The results indicate that the polysaccharide and nucleic acid region (900-1200 cm−1) is the only credible region for bacterial typing, and typing based on this region not only reduces the size of the data analysis, but results in more reliable typing results.

Chitin-Active Lytic Polysaccharide Monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze the cleavage of 1,4-glycosidic bonds various plant cell wall polysaccharides and chitin. In contrast to glycoside hydrolases, LPMOs are active on the crystalline regions of polysaccharides and thus synergize with hydrolytic enzymes. This synergism leads to an overall increase in the biomass-degradation activity of enzyme mixtures. Chitin-active LPMOs were discovered in 2010 and are currently classified in families AA10, AA11, and AA15 of the Carbohydrate-Active enZYmes database, which include LPMOs from bacteria, fungi, insects, and viruses. LPMOs have become important enzymes both industrially and scientifically and, in this chapter, we provide a brief introduction to chitin-active LPMOs including a summary of the 20+ chitin-active LPMOs that have been characterized so far. Then, we describe their structural features, catalytic mechanism, and appended carbohydrate modules. Finally, we show how chitin-active LPMOs can be used to perform chemo-enzymatic modification of chitin substrates.

Using FT-IR spectroscopy for the identification of the T. cruzi, T. rangeli, and the L. chagasi species

The cross-reaction in the diagnosis results is a serious problem, leading to an incorrect treatment and several injuries to patients. The Trypanosoma rangeli and Trypanosoma cruzi belong to the genus Trypanosoma, but the Trypanosoma rangeli is a non-pathogenic parasite to humans. While Trypanosoma cruzi is the etiological agent of Chagas' disease, which affects circa 2–3 million people and more than 6000 deaths annually in Brazil. The Leishmania chagasi causes infectious disease known as visceral leishmaniasis. This diseases have in common the crossed antigenic reaction promoted by serological tests and its differentiation is relevant for epidemiological studies and clinical practice. In this study the Fourier Transform Infrared (FT-IR) Spectroscopy was used to differentiate these microorganisms, which were cultivated and the spectra analyzed. Data analysis were performed by Gaussian curve fitting and multivariate statistical analysis. The cluster analysis have shown four specific regions to identify the microorganisms. The first three PCs of principal component analysis associated to linear discriminant were able to classify 95.6% of the parasites using cross-validation. The curve fitting method showed the quantitative differentiation among L. chagasi, T. cruzi, and T. rangeli species in the vibrational regions of polysaccharides, amide III, lipid esters, and fatty acid.

Functional Determinants of Extracellular Polymeric Substances in Membrane Biofouling: Experimental Evidence from Pure-Cultured Sludge Bacteria.

The aim of this work was to better understand the roles of extracellular polymeric substances (EPS) in membrane biofouling at the single-strain level. In the present study, a total of 23 bacterial strains were isolated from a sludge sample. The EPS extracted from pure-cultured bacteria were assessed for their fouling potentials and were simultaneously analyzed using Fourier transform infrared spectroscopy (FTIR). Further, the impact of calcium on the chemical composition of EPS and membrane fouling behavior was investigated in a strain-dependent manner. The EPS of the 23 bacterial strains exhibited different IR features for protein and polysaccharide regions. In addition, an α-1,4-glycosidic linkage (920 cm-1) and amide II (1,550 cm-1) correlated very well with the fouling potentials of all pure-cultured bacteria. In contrast to low-fouling strains, medium- and high-fouling strains exhibited two distinct peaks at 1,020 cm-1 (uronic acids) and 1,250 cm-1 (O-acetyl), which accelerate membrane fouling given their gelling capacities. In the presence of calcium, the fouling potential of a high-fouling strain (Bacillus sp. strain JSB10) was profoundly reduced (P < 0.0005) due to the binding activity of an α-1,4-glycosidic linkage and amide II with calcium. However, the impact of calcium on a low-fouling strain (Vagococcus sp. strain JSB21) was insignificant. Two-dimensional FTIR correlation spectroscopic (2D-FTIR-COS) analysis further revealed that the susceptibilities of functional groups to calcium largely relied on the composition and abundance of the above-described functional groups in EPS. These findings suggest that bacterial strains with different fouling potentials exhibit varied responses to calcium.IMPORTANCE Membrane biofouling is one of the main challenges for the operation of membrane-based processes used for water and wastewater treatment. This study revealed the functional determinants of EPS in membrane biofouling of 23 bacterial strains isolated from a full-scale membrane bioreactor (MBR) plant. We found that an α-1,4-glycosidic bond, amide II, and uronic acids of EPS significantly correlated with the fouling potentials of bacteria. The roles of these EPS groups in membrane fouling were impacted by calcium resulting from EPS-calcium interactions. In addition, our results also demonstrated that any perturbations in the sludge bacterial community in MBRs can lead to varied filtration potentials of the bulk liquor.

Interaction of Mannose-Binding Lectin With Lipopolysaccharide Outer Core Region and Its Biological Consequences.

Lipopolysaccharide (LPS, endotoxin), the main surface antigen and virulence factor of Gram-negative bacteria, is composed of lipid A, core oligosaccharide, and O-specific polysaccharide (O-PS) regions. Each LPS region is capable of complement activation. We have demonstrated that LPS of Hafnia alvei, an opportunistic human pathogen, reacts strongly with human and murine mannose-binding lectins (MBLs). Moreover, MBL–LPS interactions were detected for the majority of other Gram-negative species investigated. H. alvei was used as a model pathogen to investigate the biological consequences of these interactions. The core oligosaccharide region of H. alvei LPS was identified as the main target for human and murine MBL, especially l-glycero-d-manno-heptose (Hep) and N-acetyl-d-glucosamine (GlcNAc) residues within the outer core region. MBL-binding motifs of LPS are accessible to MBL on the surface of bacterial cells and LPS aggregates. Generally, the accessibility of outer core structures for interaction with MBL is highest during the lag phase of bacterial growth. The LPS core oligosaccharide–MBL interactions led to complement activation and also induced an anaphylactoid shock in mice. Unlike Klebsiella pneumoniae O3 LPS, robust lectin pathway activation of H. alvei LPS in vivo was mainly the result of outer core recognition by MBL; involvement of the O-PS is not necessary for anaphylactoid shock induction. Our results contribute to a better understanding of MBL–LPS interaction and may support development of therapeutic strategies against sepsis based on complement inhibition.

Nuclear magnetic resonance assessment of controlled hydrolysis of the capsular polysaccharides of Streptococcus pneumoniae serotypes 19A and 19F

ABSTRACTSeveral glycoconjugate-based vaccines are inserted nowadays in infant vaccination schedules of most countries. In an initial stage, polysaccharides are modified under controlled conditions. The assessment of polysaccharide regions available for conjugation process after the hydrolysis is crucial to predict the conjugation results for achieving the final active pharmaceutical ingredient. Two hydrolyzed capsular polysaccharides were analyzed by a heteronuclear single-quantum correlation experiment. It was adjusted for assessing 3JP-H to check the position of phosphate mono-ester residue after the hydrolysis process. All evaluated polysaccharides have been shown to remain with the anomeric position available for subsequent conjugation. The adjusted spectroscopic experiment was shown to be effective to study the hydrolysis of phosphate di-ester linked to capsular polysaccharides.

Development of PCRSeqTyping\u2014a novel molecular assay for typing of Streptococcus pneumoniae

BackgroundPrecise serotyping of pneumococci is essential for vaccine development, to better understand the pathogenicity and trends of drug resistance. Currently used conventional and molecular methods of serotyping are expensive and time-consuming, with limited coverage of serotypes. An accurate and rapid serotyping method with complete coverage of serotypes is an urgent necessity. This study describes the development and application of a novel technology that addresses this need.MethodsPolymerase chain reaction (PCR) was performed, targeting 1061 bp cpsB region, and the amplicon was subjected to sequencing. The sequence data was analyzed using the National Centre for Biotechnology Information database. For homologous strains, a second round of PCR, sequencing, and data analysis was performed targeting 10 group-specific genes located in the capsular polysaccharide region. Ninety-one pneumococcal reference strains were analyzed with PCRSeqTyping and compared with Quellung reaction using Pneumotest Kit (SSI, Denmark).ResultsA 100% correlation of PCRSeqTyping results was observed with Pneumotest results. Fifty-nine reference strains were uniquely identified in the first step of PCRSeqTyping. The remaining 32 homologous strains out of 91 were also uniquely identified in the second step.ConclusionThis study describes a PCRSeqTyping assay that is accurate and rapid, with high reproducibility. This assay is amenable for clinical testing and does not require culturing of the samples. It is a significant improvement over other methods because it covers all pneumococcal serotypes, and it has the potential for use in diagnostic laboratories and surveillance studies.