First Report on the Streptococcusgallolyticus (S. bovis Biotype I) DSM 13808 Exopolysaccharide Structure.

Streptococcus gallolyticus subspecies gallolyticus, known as Streptococcus bovis biotype I, is a facultative pathogen causing bacteraemia, infective endocarditis and sepsis that has been linked with colorectal cancer (CRC), but this correlation is still unclear. Bacterial surface structures, such as the major sugar antigens exposed to the outside of the microorganism, are potential virulence factors. One of the primary sugar antigens loosely attached to the cell surface is the biofilm component, exopolysaccharide (EPS). EPSs of S. bovis are poorly characterized molecules. Until now, only one S. macedonicus Sc136 EPS structure was known to the entire S. bovis group. The S. gallolyticus DSM 13808 EPS was investigated by chemical analysis, mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. The hexasaccharide repeating unit of the EPS, containing four Glc, two Rha residues and one phosphate group, has been described " →6)-α-d-Glcp-(1→3)-ß-l-Rhap-(1→4)-ß-d-Glcp-(1→3)-[ß-d-Glcp-(1→2)]-α-l-Rhap-(1→2)-α-d-Glcp-(1→P→".

A New Look at the Enterobacterial Common Antigen Forms Obtained during Rough Lipopolysaccharides Purification.

Enterobacterial common antigen (ECA) is a conserved antigen expressed by enterobacteria. It is built by trisaccharide repeating units: →3)-α-D-Fucp4NAc-(1→4)-ß-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→ and occurs in three forms: as surface-bound linear polysaccharides linked to a phosphoglyceride (ECAPG) or lipopolysaccharide - endotoxin (ECALPS), and cyclic form (ECACYC). ECA maintains, outer membrane integrity, immunogenicity, and viability of enterobacteria. A supernatant obtained after LPS ultracentrifugation was reported as a source for ECA isolation, but it has never been assessed for detailed composition besides ECACYC. We used mild acid hydrolysis and gel filtration, or zwitterionic-hydrophilic interaction liquid (ZIC®HILIC) chromatography combined with mass spectrometry for purification, fractionation, and structural analysis of rough Shigella sonnei and Escherichia coli R1 and K12 crude LPS preparations. Presented work is the first report concerning complex characteristic of all ECA forms present in LPS-derived supernatants. We demonstrated high heterogeneity of the supernatant-derived ECA that contaminate LPS purified by ultracentrifugation. Not only previously reported O-acetylated tetrameric, pentameric, and hexameric ECACYC have been identified, but also devoid of lipid moiety linear ECA built from 7 to 11 repeating units. Described results were common for all selected strains. The origin of linear ECA is discussed against the current knowledge about ECAPG and ECALPS.

The Mutation in wbaP cps Gene Cluster Selected by Phage-Borne Depolymerase Abolishes Capsule Production and Diminishes the Virulence of Klebsiella pneumoniae.

Klebsiella pneumoniae is considered one of the most critical multidrug-resistant pathogens and urgently requires new therapeutic strategies. Capsular polysaccharides (CPS), lipopolysaccharides (LPS), and exopolysaccharides (EPS) are the major virulence factors protecting K. pneumoniae against the immune response and thus may be targeted by phage-based therapeutics such as polysaccharides-degrading enzymes. Since the emergence of resistance to antibacterials is generally considered undesirable, in this study, the genetic and phenotypic characteristics of resistance to the phage-borne CPS-degrading depolymerase and its effect on K. pneumoniae virulence were investigated. The K63 serotype targeting depolymerase (KP36gp50) derived from Klebsiella siphovirus KP36 was used as the selective agent during the treatment of K. pneumoniae 486 biofilm. Genome-driven examination combined with the surface polysaccharide structural analysis of resistant mutant showed the point mutation and frameshift in the wbaP gene located within the cps gene cluster, resulting in the loss of the capsule. The sharp decline in the yield of CPS was accompanied by the production of a larger amount of smooth LPS. The modification of the surface polysaccharide layers did not affect bacterial fitness nor the insensitivity to serum complement; however, it made bacteria more prone to phagocytosis combined with the higher adherence and internalization to human lung epithelial cells. In that context, it was showed that the emerging resistance to the antivirulence agent (phage-borne capsule depolymerase) results in beneficial consequences, i.e., the sensitization to the innate immune response.

Lipopolysaccharide-Linked Enterobacterial Common Antigen (ECALPS) Occurs in Rough Strains of Escherichia coli R1, R2, and R4.

Enterobacterial common antigen (ECA) is a conserved surface antigen characteristic for Enterobacteriaceae. It is consisting of trisaccharide repeating unit, →3)-α-d-Fucp4NAc-(1→4)-ß-d-ManpNAcA-(1→4)-α-d-GlcpNAc-(1→, where prevailing forms include ECA linked to phosphatidylglycerol (ECAPG) and cyclic ECA (ECACYC). Lipopolysaccharide (LPS)-associated form (ECALPS) has been proved to date only for rough Shigella sonnei phase II. Depending on the structure organization, ECA constitutes surface antigen (ECAPG and ECALPS) or maintains the outer membrane permeability barrier (ECACYC). The existence of LPS was hypothesized in the 1960-80s on the basis of serological observations. Only a few Escherichia coli strains (i.e., R1, R2, R3, R4, and K-12) have led to the generation of anti-ECA antibodies upon immunization, excluding ECAPG as an immunogen and conjecturing ECALPS as the only immunogenic form. Here, we presented a structural survey of ECALPS in E. coli R1, R2, R3, and R4 to correlate previous serological observations with the presence of ECALPS. The low yields of ECALPS were identified in the R1, R2, and R4 strains, where ECA occupied outer core residues of LPS that used to be substituted by O-specific polysaccharide in the case of smooth LPS. Previously published observations and hypotheses regarding the immunogenicity and biosynthesis of ECALPS were discussed and correlated with presented herein structural data.

Structural Studies of the Lipopolysaccharide Isolated from Plesiomonas shigelloides O22:H3 (CNCTC 90/89).

Plesiomonas shigelloides is a Gram-negative, rod-shaped bacterium which causes foodborne intestinal infections, including gastroenteritis. It is one of the most frequent causes of travellers' diarrhoea. Lipopolysaccharide (LPS, endotoxin), an important virulence factor of the species, is in most cases characterised by a smooth character, demonstrated by the presence of all regions, such as lipid A, core oligosaccharide, and O-specific polysaccharide, where the latter part determines O-serotype. P. shigelloides LPS is still a poorly characterised virulence factor considering a "translation" of the particular O-serotype into chemical structure. To date, LPS structure has only been elucidated for 15 strains out of 102 O-serotypes. Structures of the new O-specific polysaccharide and core oligosaccharide of P. shigelloides from the Czechoslovak National Collection of Type Cultures CNCTC 90/89 LPS (O22), investigated by chemical analysis, mass spectrometry, and 1H,13C nuclear magnetic resonance (NMR) spectroscopy, have now been reported. The pentasaccharide repeating unit of the O-specific polysaccharide is built of one d-QuipNAc and is rich in four d-GalpNAcAN residues. Moreover, the new core oligosaccharide shares common features of other P. shigelloides endotoxins, i.e., the lack of phosphate groups and the presence of uronic acids.

The Impact of Insertion Sequences on O-Serotype Phenotype and Its O-Locus-Based Prediction in Klebsiella pneumoniae O2 and O1.

Klebsiella pneumoniae is a nosocomial pathogen, pointed out by the World Helth Organisation (WHO) as "critical" regarding the highly limited options of treatment. Lipopolysaccharide (LPS, O-antigen) and capsular polysaccharide (K-antigen) are its virulence factors and surface antigens, determining O- and K-serotypes and encoded by O- or K-loci. They are promising targets for antibody-based therapies (vaccines and passive immunization) as an alternative to antibiotics. To make such immunotherapy effective, knowledge about O/K-antigen structures, drift, and distribution among clinical isolates is needed. At present, the structural analysis of O-antigens is efficiently supported by bioinformatics. O- and K-loci-based genotyping by polymerase chain reaction (PCR) or whole genome sequencing WGS has been proposed as a diagnostic tool, including the Kaptive tool available in the public domain. We analyzed discrepancies for O2 serotyping between Kaptive-based predictions (O2 variant 2 serotype) and the actual phenotype (O2 variant 1) for two K. pneumoniae clinical isolates. Identified length discrepancies from the reference O-locus resulted from insertion sequences (ISs) within rfb regions of the O-loci. In silico analysis of 8130 O1 and O2 genomes available in public databases indicated a broader distribution of ISs in rfbs that may influence the actual O-antigen structure. Our results show that current high-throughput genotyping algorithms need to be further refined to consider the effects of ISs on the LPS O-serotype.

Changes in the lipopolysaccharide of Proteus mirabilis 9B-m (O11a) clinical strain in response to planktonic or biofilm type of growth.

The impact of planktonic and biofilm lifestyles of the clinical isolate Proteus mirabilis 9B-m on its lipopolysaccharide (O-polysaccharide, core region, and lipid A) was evaluated. Proteus mirabilis bacteria are able to form biofilm and lipopolysaccharide is one of the factors involved in the biofilm formation. Lipopolysaccharide was isolated from planktonic and biofilm cells of the investigated strain and analyzed by SDS-PAGE with silver staining, Western blotting and ELISA, as well as NMR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry techniques. Chemical and NMR spectroscopic analyses revealed that the structure of the O-polysaccharide of P. mirabilis 9B-m strain did not depend on the form of cell growth, but the full-length chains of the O-antigen were reduced when bacteria grew in biofilm. The study also revealed structural modifications of the core region in the lipopolysaccharide of biofilm-associated cells-peaks assigned to compounds absent in cells from the planktonic culture and not previously detected in any of the known Proteus core oligosaccharides. No differences in the lipid A structure were observed. In summary, our study demonstrated for the first time that changes in the lifestyle of P. mirabilis bacteria leads to the modifications of their important virulence factor-lipopolysaccharide.

The New Structure of Core Oligosaccharide Presented by Proteus penneri 40A and 41 Lipopolysaccharides.

The new type of core oligosaccharide in Proteus penneri 40A and 41 lipopolysaccharides has been investigated by ¹H and 13C NMR spectroscopy, electrospray ionization mass spectrometry and chemical methods. Core oligosaccharides of both strains were chosen for structural analysis based on the reactivity of LPSs with serum against P. penneri 40A core oligosaccharide-diphtheria toxoid conjugate. Structural analyses revealed that P. penneri 40A and 41 LPSs possess an identical core oligosaccharide.

The Complete Structure of the Core Oligosaccharide from Edwardsiella tarda EIB 202 Lipopolysaccharide.

The chemical structure and genomics of the lipopolysaccharide (LPS) core oligosaccharide of pathogenic Edwardsiella tarda strain EIB 202 were studied for the first time. The complete gene assignment for all LPS core biosynthesis gene functions was acquired. The complete structure of core oligosaccharide was investigated by ¹H and 13C nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry MSn, and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry. The following structure of the undecasaccharide was established: The heterogeneous appearance of the core oligosaccharide structure was due to the partial lack of ß-d-Galp and the replacement of α-d-GlcpNAcGly by α-d-GlcpNGly. The glycine location was identified by mass spectrometry.

Ficolin-2 and ficolin-3 in women with malignant and benign ovarian tumours.

Ficolins are serum pattern recognition molecules. They have opsonic properties and are able to activate complement via the lectin pathway. This paper reports investigations concerning ficolin-2 and ficolin-3 in ovarian cancer (OC). Their serum levels, single nucleotide polymorphisms of the corresponding FCN2 and FCN3 genes and specific mRNA expression in ovarian sections were investigated in 128 patients suffering from primary OC and 197 controls operated on for reasons other than malignancies. The latter consisted of two reference groups: those with benign tumours (n = 123) and those with normal ovaries (NO) (n = 74). Serum ficolin-2 and ficolin-3 concentrations were higher among patients with malignant disease when compared with either of the reference groups. A significant correlation between ficolin-2 and ficolin-3 concentrations was found, while no correlations with CA125 antigen or CRP were observed. No differences in the frequency of single nucleotide polymorphisms at sites -64, -4 (promoter), +6359, or +6424 (exon 8) (FCN2 gene) nor in the frame-shift mutation 1637delC (FCN3 gene) were found between investigated groups. In contrast to serum concentrations, the expression of FCN2 gene (reported for the first time in ovarian sections) was significantly lower in women with OC in comparison with patients with NO but not with benign ovarian tumours. In case of FCN3 gene, its expression levels in OC group inversely correlated with serum ficolin-3 and were lower in comparison with controls.

Core oligosaccharide of Plesiomonas shigelloides PCM 2231 (Serotype O17) lipopolysaccharide--structural and serological analysis.

The herein presented complete structure of the core oligosaccharide of lipopolysaccharide (LPS) P. shigelloides Polish Collection of Microorganisms (PCM) 2231 (serotype O17) was investigated by (1)H, (13)C NMR spectroscopy, mass spectrometry, chemical analyses and serological methods. The core oligosaccharide is composed of an undecasaccharide, which represents the second core type identified for P. shigelloides serotype O17 LPS. This structure is similar to that of the core oligosaccharide of P. shigelloides strains 302-73 (serotype O1) and 7-63 (serotype O17) and differs from these only by one sugar residue. Serological screening of 55 strains of P. shigelloides with the use of serum against identified core oligosaccharide conjugated with bovine serum albumin (BSA) indicated the presence of similar structures in the LPS core region of 28 O-serotypes. This observation suggests that the core oligosaccharide structure present in strain PCM 2231 could be the most common type among P. shigelloides lipopolysaccharides.

New functional ligands for ficolin-3 among lipopolysaccharides of Hafnia alvei.

Ficolin-1 (M), ficolin-2 (L), ficolin-3 (H) and mannan-binding lectin (MBL) activate the complement system and have opsonic activity. The specificity of ficolin-3 is poorly characterized and currently limited to a few ligands only. We present new specific targets for human ficolin-3, identified among lipopolysaccharides (LPSs, endotoxin) of Hafnia alvei. The interaction was restricted to LPSs of four strains: 23, Polish Collection of Microorganisms (PCM) 1200, PCM 1203 and PCM 1205 and limited to their O-specific polysaccharides (O-specific PSs) composed of different numbers of oligosaccharide (OS) repeating units (RUs). Moreover, these LPS/ficolin-3 complexes activated the lectin pathway of complement in a C4b-deposition assay in a calcium- and magnesium-dependent way. A neoglycoconjugate of the O-specific PS fraction of H. alvei 1200 LPS with bovine serum albumin (BSA) was prepared and used as a tool for the determination of ficolin-3 concentration and activity in serum. To confirm a structure of the O-specific PS 1200 selected for the conjugate preparation, structural analysis was performed on a series of O-specific PSs released by the mild acid hydrolysis of the LPS. The isolated O-specific PSs, showing the different length distributions, were devoid of a major part of the core OS region and had Hep-Kdo disaccharide at a reducing end. The neoglycoconjugate was a highly selective tool for the determination of ficolin-3 concentration and activity in serum (lectin pathway activation in the C4b deposition assay) and was not affected by MBL, ficolin-1 and ficolin-2 or natural antibodies.

Synoptic CT scan reporting of pancreatic adenocarcinoma to align with international consensus guidelines on surgical resectability: a Victorian pilot.

BACKGROUND: Non-metastatic pancreatic ductal adenocarcinoma (PDAC) is classified as resectable (R), borderline resectable (BR) or locally advanced (LA). International Consensus Guidelines on these definitions exist, but have not been integrated into everyday Australian practice. The anatomical features on CT imaging lend themselves to synoptic reporting which should enhance completeness, comparability and consistency. METHODS: We developed and tested a synoptic report for PDAC derived from the International Consensus Guidelines at two metropolitan pancreatic cancer services to standardize CT reporting in the region. Consecutive scans with suspected PDAC discussed at multidisciplinary meetings were reported using the template between October 2020 and September 2021. A purpose-built database captured data regarding resectability and image-quality parameters. RESULTS: Ninety-five scans were reviewed, 57.9% (N = 55) of which conformed to high-quality pancreatic CT protocols. Of suboptimal scans, meaningful synoptic reports were able to be issued for a further 24/40 (due to metastases in 9, and unequivocal resectability status in 15). Of 79 classifiable scans, 20% were metastatic, 51% deemed resectable, 16% locally advanced and 13% borderline resectable. DISCUSSION: PDAC lends itself to synoptic reporting given the specific anatomical considerations that classify resectability. This relies, however, on high-quality CT imaging and it was surprising that over 40% of scans reviewed were of suboptimal quality. Despite this, resectability status according to the International Consensus Guidelines was designated for 83% of scans. Optimal treatment algorithms for LA, BR and resectable disease vary widely underscoring the critical importance of accurately differentiating these anatomic subtypes of PDAC, and thus support further implementation of a synoptic report of this nature.

Structures of two novel, serologically nonrelated core oligosaccharides of Yokenella regensburgei lipopolysaccharides differing only by a single hexose substitution.

Immunochemical analysis of the Yokenella regensburgei lipopolysaccharides (LPS) indicated the presence of the core oligosaccharide-related immunotypes among the investigated strains. The structure of the core oligosaccharide segment of the Y. regensburgei LPS has been investigated using chemical methods, mass spectrometry, and (1)H, (13)C NMR spectroscopy. It was concluded that the core oligosaccharides of the strains PCM 2476 and PCM 2477 are composed of an undecasaccharide. The combined data revealed two immunotypes of the core oligosaccharide recognized by antibodies against the whole bacterial cells. The structural differences between the core oligosaccharides are limited to the outermost terminal hexopyranose residue. In the core oligosaccharide of the strain PCM 2476, it was identified as alpha-d-Glcp and in that of the strain PCM 2477 as alpha-d-Galp. This subtle difference between the glycoforms of the LPS core appeared to be essential for formation of the epitopes recognized by the specific antibodies directed against the Y. regensburgei whole bacterial cells. The oligosaccharides are not substituted by phosphate groups. Instead, the carboxyl groups of Kdo and galacturonic acid residues present in the core provide the negative charges. The undecasaccharides represent a novel core type of bacterial LPS, which is characteristic for Y. regensburgei.

De­O­acylated lipooligosaccharide of E. coli B reduces the number of metastatic foci via downregulation of myeloid cell activity.

Lipopolysaccharides are the main surface antigens and virulence factors of gram­negative bacteria. Removal of four ester­bound fatty acid residues from hexaacyl lipid A of Escherichia coli lipooligosaccharide (LOS) resulted in the de­O­acylated derivative E. coli LOS­OH (LOS­OH). This procedure caused a significant reduction in the toxicity of this compound compared to the native molecule. We investigated the effect of such a structural LOS modification on its biological activity using in vitro assays with monocytic cells of the RAW264.7 line, dendritic cells of the JAWS II line, bone marrow­derived dendritic cells (BM­DCs), and spleen cells. Furthermore, in in vivo experiments with a melanoma B16 metastasis model, the anti­metastatic activity of the compounds and spleen cell reactivity mediated by them representing a systemic response were analyzed. The results revealed that LOS­OH demonstrated weaker ability than LOS to stimulate and polarize an immune response both in vitro and in vivo. It induced lower cytokine production by cells of myeloid lines. Multiple applications of LOS­OH into mice injected intravenously with B16 cells significantly (P<0.05; P<0.01) reduced the number of metastatic foci in the lungs, presumably via silencing of myeloid cell reactivity as well as the inability to stimulate lymphoid cells both directly and indirectly. These findings suggest that LOS­OH maintained in the body of metastasis­bearing mice appears to modulate or downregulate the innate response, leading to the inability of blood myeloid cells to support the migration of melanoma cells to lung tissue.

Interactions of ficolin-3 with ovarian cancer cells.

BACKGROUND: Ficolin-3 is a pattern-recognition molecule with the ability to activate the lectin pathway of complement. It is found in lung, liver and blood, but its physiological role is unclear. We have investigated interaction of recombinant ficolin-3 with malignant cells and tissues. MATERIAL AND METHODS: Cells of various lines of human origin as well as ovarian tissue sections have been studied with the use of flow cytometry and immunohistochemistry. RESULTS: Recombinant (but not serum-derived) ficolin-3 was found to bind strongly to the ovarian cancer cell lines, SKOV-3, OVCAR-3 and ES-2, at concentrations of 2.5 µg/ml and above. Moreover, His-tagged recombinant ficolin-3 (10 µg/ml) preferentially stained ovarian tissue sections from patients with malignant tumours compared with those from patients without. Binding to cell lines was inhibited by EDTA and specific carbohydrate ligands, indicating involvement of the fibrinogen-like domain. Binding was enhanced under mildly acidic conditions and at physiological pH after pre-incubation of cells with mildly acidic buffer. CONCLUSION: Basing on data concerning recombinant protein, it may be suggested that ficolin-3 is involved in immune response in ovarian cancer. However, unidentified serum factor(s) seem(s) to protect cancer cells from recognition by natural or rficolin-3.

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.

A New Ligand-Based Method for Purifying Active Human Plasma-Derived Ficolin-3 Complexes Supports the Phenomenon of Crosstalk between Pattern-Recognition Molecules and Immunoglobulins.

Despite recombinant protein technology development, proteins isolated from natural sources remain important for structure and activity determination. Ficolins represent a class of proteins that are difficult to isolate. To date, three methods for purifying ficolin-3 from plasma/serum have been proposed, defined by most critical step: (i) hydroxyapatite absorption chromatography (ii) N-acetylated human serum albumin affinity chromatography and (iii) anti-ficolin-3 monoclonal antibody-based affinity chromatography. We present a new protocol for purifying ficolin-3 complexes from human plasma that is based on an exclusive ligand: the O-specific polysaccharide of Hafnia alvei PCM 1200 LPS (O-PS 1200). The protocol includes (i) poly(ethylene glycol) precipitation; (ii) yeast and l-fucose incubation, for depletion of mannose-binding lectin; (iii) affinity chromatography using O-PS 1200-Sepharose; (iv) size-exclusion chromatography. Application of this protocol yielded average 2.2 mg of ficolin-3 preparation free of mannose-binding lectin (MBL), ficolin-1 and -2 from 500 ml of plasma. The protein was complexed with MBL-associated serine proteases (MASPs) and was able to activate the complement in vitro. In-process monitoring of MBL, ficolins, and total protein content revealed the presence of difficult-to-remove immunoglobulin G, M and A, in some extent in agreement with recent findings suggesting crosstalk between IgG and ficolin-3. We demonstrated that recombinant ficolin-3 interacts with IgG and IgM in a concentration-dependent manner. Although this association does not appear to influence ficolin-3-ligand interactions in vitro, it may have numerous consequences in vivo. Thus our purification procedure provides Ig-ficolin-3/MASP complexes that might be useful for gaining further insight into the crosstalk and biological activity of ficolin-3.

Fractionation and analysis of lipopolysaccharide-derived oligosaccharides by zwitterionic-type hydrophilic interaction liquid chromatography coupled with electrospray ionisation mass spectrometry.

Lipopolysaccharide (LPS, endotoxin) is a main surface antigen and virulence factor of Gram-negative bacteria. Regardless of the source of LPS, this molecule, isolated from the smooth forms of bacteria, is characterised by a general structural layout encompassing three regions: (i) an O-specific polysaccharide (O-PS) - a polymer of repeating oligosaccharide units, (ii) core oligosaccharide (OS), and (iii) the lipid A anchoring LPS in the outer membrane of the cell envelope of Gram-negative bacteria. Structural analysis usually requires degradation of LPS and further efficient separation of various poly- and oligosaccharide glycoforms. The hydrophilic interaction liquid chromatography (HILIC) was shown as an efficient technique for separation of labelled or native neutral and acidic glycans, glycopeptides, sialylated glycans, glycosylated and nonglycosylated peptides. Herein we adopted ZIC(®) (zwitterionic stationary phase covalently attached to porous silica)-HILIC technology in combination with electrospray ionisation mass spectrometry to separate different LPS-derived oligosaccharides. As a result three effective procedures have been developed: (i) to separate different core oligosaccharides of Escherichia coli R1 LOS, (ii) to separate RU-[Hep]-Kdo oligosaccharides from core OS glycoforms of Hafnia alvei PCM 1200 LPS, and (iii) to separate Hep and Kdo-containing mono, di-, tri- and tetrasaccharides of H. alvei PCM 1200 LPS. Moreover, some of developed analytical procedures were scaled to semi-preparative protocols and used to obtain highly-purified fractions of the interest in larger quantities required for future evaluation, analysis, and biological applications.

Core oligosaccharide of Escherichia coli B-the structure required for bacteriophage T4 recognition.

The structure of Escherichia coli B strain PCM 1935 core oligosaccharide has been investigated by (1)H and (13)C NMR spectroscopy, MALDI-TOF MS and ESI MS(n). It was concluded that the core oligosaccharide is a pentasaccharide with the following structure: ESI MS/MS analysis revealed that the glycine (a minor component) is linked to the →3,7)-l-α-d-Hepp-(1→ residue.