Chemoenzymatic synthesis of an α-1,6-glucan-based conjugate vaccine against Helicobacter pylori.

In this study, we investigated the utility of glycoconjugates based on a linear α-1,6-glucan chain synthesized using a recombinant α-1,6-glucosyltransferase from the 26695 strain of Helicobacter pylori. Capillary electrophoresis-mass spectrometry analysis confirmed the main product to contain 9-10 sequentially added α-1,6-linked glucose residues. This was consistent with a length of α-1,6-glucan structure present in the outer core region of H. pylori lipopolysaccharide (LPS) from strains 26695 and 26695 HP0826::Kan. The synthetic α-1,6-glucan was conjugated to either bovine serum albumin or tetanus toxoid and immunological properties of resultant glycoconjugates investigated. The conjugates were immunogenic in rabbits and mice and induced strong and specific IgG responses against purified LPS from typeable and nontypeable α-1,6-glucan-positive H. pylori strains. Furthermore, the post-immune sera from rabbits that received the conjugates were bactericidal and cross-reacted with selected clarithromycin-resistant and clarithromycin-susceptible clinical isolates of H. pylori. This technology offers a novel approach to the design of a synthetic carbohydrate-based vaccine against H. pylori.

Structural investigation of the capsular polysaccharide from a clinical isolate of Fusobacterium necrophorum subspecies necrophorum biotype a strain LA 81-617.

A capsular polysaccharide (CPS) from Fusobacterium necrophorum subspecies necrophorum biotype A strain LA 81-617 was isolated from a saline cell wash and purified by gel and anion-exchange chromatography. The structure of the CPS was studied by one- and two-dimensional 1H and 13C NMR spectroscopy techniques in combination with electrospray ionization mass spectrometry (ESI-MS). The CPS was found to resemble the bacterial cell-wall murein polysaccharide backbone, consisting of N-acetylglucosamine and N-acetylmuramic acid (MurNAc) components, with the following structure:-[-4-ß-MurNAc-4-ß-GlcNAc-]n-with N-acetyl-1,6-anhydro-ß-muramic acid at the reducing end. This is the first report on the structure of F. necrophorum capsular polysaccharide.

Structural and immunological characterization of a glycoconjugate based on the delipidated lipopolysaccharide from a nontypeable Helicobacter pylori strain PJ1 containing an extended d-glycero-d-manno-heptan.

Structural characterization of the lipopolysaccharide (LPS) from a nontypeable Helicobacter pylori strain PJ1 and two corresponding mutants, PJ1 HP1283:cam and PJ1 HP1284:cam, was performed using a combination of NMR and mass spectrometric techniques. It resulted in the core structure that differed significantly from the one proposed previously. Overall architecture of PJ1 LPS was found to be consistent with a structural model described for several other H. pylori strains. It contained a polymer of d-glycero-d-manno-heptose (dd-Hep) as the O-chain component, linked to α-1,6-glucan through a dd-Hep oligosaccharide. H. pylori PJ1 HP1283:cam LPS was missing dd-heptan, terminating with an α-1,6-glucan chain containing 5-13 glucose residues. LPS of strain PJ1 HP1284:cam was missing dd-Hep from the core and had ß-GlcNAc attached directly to O-3 of the inner-core ld-Hep residue. To investigate the role of dd-heptan in protective immunity, delipidated LPS (dLPS) from strain PJ1 was conjugated to tetanus toxoid (TT) and immunological properties of the resultant glycoconjugate dLPS(PJ1)-TT determined. The dLPS(PJ1)-TT conjugate was immunogenic in mice and rabbits and induced specific and cross-reactive functional antibodies against homologous and heterologous strains of H. pylori. Whole cell indirect ELISA performed on a selected number of H. pylori isolates confirmed that the immune response correlated with the presence of α-1,6-glucan and was not augmented by the dd-Hep content of these strains.

Antimicrobial susceptibility of Canadian isolates of Helicobacter pylori in Northeastern Ontario.

BACKGROUND: Helicobacter pylori plays a significant role in gastritis and ulcers. It is a carcinogen as defined by the WHO, and infection can result in adenocarcinomas and mucosa-associated lymphoid tissue lymphomas. In Canada, rates of antimicrobial resistance are relatively unknown, with very few studies conducted in the past 15 years. OBJECTIVE: To examine rates of resistance in Sudbury, Ontario, compare antimicrobial susceptibility methods and attempt to determine the molecular basis of antibiotic resistance. METHODS: Patients attending scheduled visits at Health Sciences North (Sudbury, Ontario) provided gastric biopsy samples on a volunteer basis. In total, 20 H pylori isolates were collected, and antimicrobial susceptibility testing (on amoxicillin, tetracycline, metronidazole, ciprofloxacin, levofloxacin and clarithromycin) was conducted using disk diffusion and E-test methods. Subsequently, genomic DNA from these isolates was sequenced to detect mutations associated with antimicrobial resistance. RESULTS: Sixty-five percent of the isolates were found to be resistant to at least one of the listed antibiotics according to E-test. Three isolates were found to be resistant to ≥3 of the above-mentioned antibiotics. Notably, 25% of the isolates were found to be resistant to both metronidazole and clarithromycin, two antibiotics that are normally prescribed as part of first-line regimens in the treatment of H pylori infections in Canada and most of the world. Among the resistant strains, the sequences of 23S ribosomal RNA and gyrA, which are linked to clarithromycin and ciprofloxacin/levofloxacin resistance, respectively, revealed the presence of known point mutations associated with antimicrobial resistance. CONCLUSIONS: In general, resistance to metronidazole, ciprofloxacin/levofloxacin and clarithromycin has increased since the studies in the early 2000s. These results suggest that surveillance programs of H pylori antibiotic resistance may need to be revisited or improved to prevent antimicrobial therapy failure.

HISTORIQUE: L'Helicobacter pylori contribue énormément à la gastrite et aux ulcères. L'OMS le définit comme un cancérigène, et l'infection peut provoquer l'apparition d'adénocarcinomes et de lymphomes de tissus lymphoïdes associés aux muqueuses. Au Canada, on connaît relativement peu les taux de résistance antimicrobienne, car très peu d'études ont été réalisées sur le sujet depuis 15 ans. OBJECTIF: Examiner les taux de résistance à Sudbury, en Ontario, comparer les méthodes de susceptibilité antimicrobienne et tenter de déterminer le fondement biologique de la résistance antibiotique. MÉTHODOLOGIE: Les patients qui allaient à un rendez-vous prévu au Health Sciences North de Sudbury ont remis les résultats de biopsies gastriques sur une base volontaire. Au total, 20 isolats de H pylori ont été recueillis, et les tests de susceptibilité antimicrobienne (à l'amoxicilline, à la tétracycline, au métronidazole, à la ciprofloxacine, à la lévofloxacine et à la clarithromycine) ont été effectués au moyen de la diffusion sur disque et de l'essai E. L'ADN génomique de ces isolats a ensuite été séquencé pour déceler les mutations associées à la résistance antimicrobienne. RÉSULTATS: Selon l'essai E, 65 % des isolats étaient résistants à au moins un des antibiotiques énumérés. Notamment, 25 % des isolats étaient résistants à la fois au métronidazole et à la clarithromycine, tous deux normalement prescrits en première ligne pour traiter les infections à H pylori au Canada et dans la plupart des régions du monde. Parmi les souches résistantes, les séquences d'ARN ribosomique 23S et de gyrA, qui sont liées à la résistance à la clarithromycine et à la ciprofloxacine-lévofloxacine, respectivement, révélaient la présence de mutations de points connus associés à une résistance antimicrobienne. CONCLUSIONS: En général, la résistance au métronidazole, à la ciprofloxacine-lévofloxacine et à la clarithromycine a augmenté depuis les études réalisées au début des années 2000. D'après ces résultats, il faudra peut-être revoir ou améliorer les programmes de surveillance de l'antibiorésistance au H pylori pour prévenir l'échec du traitement antimicrobien.

Lipopolysaccharide glycotyping of clarithromycin-resistant and clarithromycin-susceptible Canadian isolates of Helicobacter pylori.

Lipopolysaccharide (LPS) of Helicobacter pylori exhibits several unique structures, such as Lewis (Le) antigens, α-1,6-glucan, and dd-heptan. To investigate the relationship between LPS structure and resistance to clarithromycin, 41 Canadian isolates of H. pylori were characterized by whole-cell ELISA (enzyme-linked immunosorbent assay), sugar analysis, immunoblotting, and indirect immunofluorescence. The expression of type 2 Lewis X and (or) Lewis Y antigens was detected in 22 of 23 (95.7%) clarithromycin-resistant and in 14 of 18 (77.7%) clarithromycin-susceptible H. pylori strains (P < 0.05), and 8 isolates co-expressed type 1 and type 2 Le antigens (8/41, 19.5%). A significantly higher frequency of α-1,6-glucan (P < 0.01) was detected in clarithromycin-resistant strains than in clarithromycin-susceptible strains (19/23 (82.6%) versus 11/18 (61.1%)). Sugar analysis of selected α-1,6-glucan-positive H. pylori strains confirmed that they frequently contained elevated amounts of dd-heptose. Clarithromycin-resistant isolates were also characterized by low expression levels or absence of CagA (17/23, 73.9%). Indirect immunofluorescence studies carried out on selected H. pylori strains with rabbit immune sera specific for α-1,6-glucan confirmed broad recognition of α-1,6-glucan epitope. The binding was not affected by LPS glycotype of H. pylori isolates examined nor by their CagA status or resistance to clarithromycin. These findings suggest α-1,6-glucan as a potential vaccine target, especially in an era of increasing clarithromycin resistance in H. pylori.

The potential of dextran-based glycoconjugates for development of Helicobacter pylori vaccine.

We have recently demonstrated that synthetic glycoconjugates based on delipidated lipopolysaccharide (LPS) of Helicobacter pylori and containing an α(1-6)-glucan chain induced broadly cross-reactive functional antibodies in immunized animals. To investigate the candidacy of α(1-6)-glucan as an alternative vaccine strategy we prepared glycoconjugates based on dextrans produced by lactic acid bacteria Leuconostoc mesenteroides B512F and consisting of linear α(1-6)-glucan chains with limited branching. Three dextrans with averaged molecular masses of 5,000 Da, 3,500 Da and 1,500 Da, respectively, were modified with a diamino group-containing linker and conjugated to a carrier protein, tetanus toxoid (TT) or diphtheria toxoid (DT), and their immunological properties investigated. The conjugates were immunogenic in both rabbits and mice and induced specific IgG responses against α(1-6)-glucan-expressing H. pylori LPS. Studies performed with post-immune sera of mice and rabbits immunized with dextran-based conjugates demonstrated cross-reactivity with LPS from typeable and non-typeable strains of H. pylori and selected mutants. The post-immune sera from rabbits that received the conjugates exhibited functional activity against α(1-6)-glucan-positive strains of H. pylori. These data provide evidence that dextran-based conjugates may offer a simplified approach to the development of carbohydrate-based vaccines against H. pylori.

Colonization of gerbils with Helicobacter pylori O-chain-deficient mutant SS1 HP0826::Kan results in gastritis and is associated with de novo synthesis of extended homopolymers.

The O-chain polysaccharide of Helicobacter pylori is important for colonization and generation of chronic gastritis in mice. There are marked host differences in the development of H. pylori-induced gastric pathology in mice and gerbils. To investigate the role of the O-chain polysaccharide of H. pylori in colonization and gastritis in Mongolian gerbils, inoculation by oral gavage with H. pylori strain SS1 and its corresponding O-chain polysaccharide-deficient mutant SS1 HP0826::Kan was undertaken. Infection with both strains resulted in corpus atrophy, loss of parietal cells, and extensive mucous metaplasia at both 18 and 30 weeks postinfection. Contrary to previous results in splenocyte recipient severe combined immunodeficiency (SCID) mice, no difference was found in the grade of chronic gastritis, polymorphonuclear cell infiltration, atrophy, and mucous metaplasia in gerbils infected with the wild-type SS1 strain or SS1 HP0826::Kan strain. Examination of the effects of gerbil passage on LPS profiles of output SS1 HP0826::Kan isolates by SDS-PAGE, sugar, and methylation analyses revealed significant differences in LPS profiles of SS1 HP0826::Kan cells recovered from infected gerbils as compared to input bacteria. Specifically, the presence of a novel homopolymer of d-galactan, as well as an extended polymer of d-riban, was detected. These data provide evidence for the role of H. pylori LPS in bacterial adaption to promote colonization and pathology.

Lipopolysaccharide structure of Helicobacter pylori serogroup O:3.

In this study, we describe a re-investigation of the lipopolysaccharide structure of Helicobacter pylori serogroup O:3. Application of NMR and MS approaches to the analysis of oligosaccharides obtained through degradation of LPS from H. pylori serogroup O:3 by various methods confirmed that its general architecture was identical to that of LPS from H. pylori strains 26695 and SS1 and followed a sequential linear assembly of the α-1,6-glucan, dd-heptan, and O-chain components. Additionally, MALDI-MS analysis demonstrated that a significant proportion of H. pylori serogroup O:3 LPS was terminated with α-1,6-glucan and was not further substituted by dd-heptan and the O-chain polysaccharide.

Neglected infectious diseases in Aboriginal communities: Haemophilus influenzae serotype a and Helicobacter pylori.

This report describes proceedings of a workshop entitled "Neglected Infectious Diseases in Aboriginal Communities" which took place in Thunder Bay, Ontario, Canada, on October 12, 2011. This workshop was jointly organized by the National Research Council of Canada (NRC), the National Microbiology Laboratory (Public Health Agency of Canada) and Northern Ontario School of Medicine (NOSM) with participants from the Medical Sciences Division and Clinical Sciences Division of NOSM, NRC, National Microbiology Laboratory (NML), Public Health Laboratory (Thunder Bay), Thunder Bay District Health Unit, and Regional Health Survey at Chiefs of Ontario. The main purpose of the workshop was to summarize the current state of knowledge on two less publicized infectious disease agents afflicting Canadian Aboriginal communities: Haemophilus influenzae serotype a (Hia) and Helicobacter pylori. Another highlight of this workshop was the discussion on novel approaches for vaccination strategies in the control and prevention of such disease agents. In conclusion, a long-term collaborative research framework was established between NRC, NML and NOSM to develop carbohydrate-based vaccines against these pathogens that may benefit the health of Canadian Aboriginal peoples and other population groups at risk.

Design and immunological properties of Helicobacter pylori glycoconjugates based on a truncated lipopolysaccharide lacking Lewis antigen and comprising an α-1,6-glucan chain.

To investigate the vaccine potential of H. pylori lipopolysaccharide (LPS), truncated LPS of H. pylori strain 26695 HP0826::Kan lacking O-chain polysaccharide and comprising an extended α-1,6-linked glucan chain was conjugated to tetanus toxoid (TT) or bovine serum albumin (BSA). Two approaches were used for delipidation or partial delipidation of H. pylori LPS: (1) mild hydrolysis resulting in delipidated LPS (dLPS) and (2) treatment with anhydrous hydrazine resulting in removal of O-linked fatty acids (LPS-OH). Both LPS-OH and dLPS were covalently linked through a 2-keto-3-deoxy-octulosonic acid (Kdo) residue to a diamino group-containing spacer, followed by conjugation to thiolated TT or BSA to give conjugates LPS-OH-TT, dLPS-BSA and dLPS-TT, respectively. The LPS-OH-TT, dLPS-BSA and dLPS-TT conjugates were immunogenic in both rabbits and mice, inducing strong and specific IgG responses against homologous and heterologous strains of H. pylori. Moreover, the rabbit post-immune sera showed cross-reactivity against clinical isolates of H. pylori in a whole-cell indirect ELISA, which was further confirmed by indirect immunofluorescent microscopy. A tenfold stronger IgG immune response to the immunizing antigen was generated in mice and rabbits that received dLPS-containing conjugate. The post-immune sera of rabbits immunized with LPS-OH-TT, dLPS-BSA or dLPS-TT displayed significant bactericidal activity against mutant and wild-type α-1,6-glucan-expressing strains and selected clinical isolates of H. pylori. Finally, partial protection against H. pylori challenge was demonstrated in mice vaccinated with dLPS-TT conjugate adjuvanted with cholera toxin. In summary, this study shows that glycoconjugates based on delipidated or partially delipidated LPS from H. pylori 26695 HP0826::Kan mutant induce broadly cross-reactive functional antibodies in immunized animals and should be considered for further vaccine development and testing.

Simultaneous analysis of cardiolipin and lipid A from Helicobacter pylori by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Cardiolipin (CL) is an anionic tetraacylphospholipid found in mammalian tissues, inner membrane of mitochondria and in the cytoplasmic membrane of Gram-positive and -negative bacteria. Lipid A is the principal structural component responsible for the range of biological activities of lipopolysaccharides. Here we report a MALDI-MS-based method for the sensitive simultaneous analysis of CL and lipid A from Helicobacter pylori cells. The sensitivity was demonstrated by the analysis of CL and lipid A from a single bacterial colony of in vitro grown H. pylori strain NCTC 11637 (ATCC 43504). We then characterized the CL and lipid A structures in H. pylori cells grown under three different conditions, on agar-horse blood plates, in liquid culture and ex vivo. The results revealed the presence of high amounts of myristic (C14:0) and 19-carbon cyclopropane (C19:0cyc) fatty acids. Alterations in CL structure were observed in H. pylori cells cultivated on plates as compared with the bacteria grown in broth culture. Furthermore, significant changes in lipid A acylation pattern were detected in H. pylori cells during formation of coccoids. In contrast, structural analysis of CL from ex vivo H. pylori cells recovered from the stomachs of infected Mongolian gerbils demonstrated only minor changes in acyl chain combination. This is the first report of simultaneous analysis of CL and lipid A from ex vivo cells of H. pylori.

Helicobacter pylori isolates from Greek children express type 2 and type 1 Lewis and α1,6-glucan antigens in conjunction with a functional type IV secretion system.

Helicobacter pylori infection is often acquired in childhood and can persist for life. Previous studies in adult patients have shown that H. pylori isolates from North American and European hosts express predominantly type 2 Lewis x (Le(x)) and Le(y) epitopes, while Asian strains have the capacity to express type 1 Le(a) and Le(b) structures. In order to understand the influence of environmental and host factors on the expression of Le antigens, we analysed 50 Greek H. pylori isolates from symptomatic children. Both CagA-positive and -negative strains were evaluated. The expression of Le antigens was determined by whole-cell indirect ELISA (WCE), and LPS profiles were assessed by gel electrophoresis and immunoblotting. Occurrence of Le(x) and/or Le(y) antigens was confirmed in 35 of the isolates (70 %) while 15 of the isolates were non-typable. It was found that 11 of the paediatric isolates had the propensity to express type 1 Le(b) blood-group antigen (22 %), a feature relatively uncommon in H. pylori isolates from adults. One strain expressed both Le(b) and Le(a) antigens. The majority of the isolates (49/50, 98 %) expressed α1,6-glucan, an antigenic non-Le determinant present in the outer core region of H. pylori LPS. All Le(x)- and Le(y)-expressing strains also carried a functional cag pathogenicity island-encoding a type IV secretion system, capable of translocating CagA protein, as well as the vacAs1 allele, suggesting that Le(x) and Le(y) epitopes may aid the persistence of more aggressive strains. No association between bacterial virulence characteristics and the histopathological observations was evident.

Characterization and functional activity of murine monoclonal antibodies specific for α1,6-glucan chain of Helicobacter pylori lipopolysaccharide.

BACKGROUND: The outer core region of H. pylori lipopolysaccharide (LPS) contains α1,6-glucan previously shown to contribute to colonizing efficiency of a mouse stomach. The aim of the present study was to generate monoclonal antibodies (mAbs) specific for α1,6-glucan and characterize their binding properties and functional activity. MATERIALS AND METHODS: BALB/c mice were injected intraperitoneally with 10(8) formalin-fixed H. pylori O:3 0826::Kan cells 3× over 56 days to achieve significant titer. Anti-α1,6-glucan-producing hybridomas were screened by indirect ELISA using purified H. pylori O:3 0826::Kan LPS. One clone, 1C4F9, was selected for further characterization. The specificities of mAbs were determined by indirect and inhibition ELISA using structurally defined H. pylori LPS and synthetic oligosaccharides, and whole-cell indirect ELISA (WCE) of clinical isolates. They were further characterized by indirect immunofluorescent (IF) microscopy and their functional activity in vitro determined by serum bactericidal assays against wild-type and mutant strains of H. pylori. RESULTS: The generated anti-α1,6-glucan IgM, 1C4F9, has demonstrated an excellent specificity for the glucan chain containing 5 to 6 α1,6-linked glucose residues and showed surface accessibility by IF microscopy with H. pylori cells adherent to gastric adenocarcinoma cells monolayers. Of 38 isolates from Chile, 17 strains reacted with antiglucan mAbs in WCE (OD450 ≥ 0.2). Bactericidal activity was observed against selective wild-type and mutant H. pylori strains exhibiting OD450 values of ≥ 0.45 in WCE. CONCLUSIONS: Anti-α1,6-glucan mAbs could have potential application in typing and surveillance of H. pylori isolates as well as offer insights into structural requirements for the development of LPS-based vaccine against H. pylori infections.

Lipopolysaccharide structures of Helicobacter pylori wild-type strain 26695 and 26695 HP0826::Kan mutant devoid of the O-chain polysaccharide component.

We describe a re-investigation of the structure of the lipopolysaccharide (LPS) from Helicobacter pylori genomic strain 26695 and its corresponding HP0826::Kan mutant lacking the O-chain component based on the in-depth NMR analysis of the oligosaccharide products obtained through the use of various degradation procedures performed on the purified LPS from both strains, as well as CE-MS data. New structural evidence indicates the presence of the linear arrangement of glucan and heptan portions of the LPS attached through -6-α-DDHep-3-α-L-Fuc-3-ß-GlcNAc- fragment to the inner core DD-heptose residue. This structure differs from previously reported structures of the H. pylori 26695 LPS in several aspects.

A reinvestigation of the lipopolysaccharide structure of Helicobacter pylori strain Sydney (SS1).

In this study, we describe a reinvestigation of the lipopolysaccharide (LPS) structure of Helicobacter pylori strain Sydney (SS1) based on the NMR analysis of oligosaccharides obtained through the use of various degradations of the LPS as well as capillary electrophoresis-MS data. The results of the analysis indicated that the core region of a major H. pylori SS1 LPS glycoform consists of a backbone core oligosaccharide substituted at the D-glycero-D-manno-heptose (DD-Hep) residue by a linear chain composed of a trisaccharide fragment α-ddHep-3-α-L-Fuc-3-ß-GlcNAc, as previously demonstrated for H. pylori strain 26695, further elongated by consecutively added α-Glc and ß-Gal residues, and terminating in a novel linear chain consisting of 1,2-linked ß-ribofuranosyl residues, where the last ß-ribofuranosyl residue provides a point of attachment for the O-chain polysaccharide: [Formula: see text] where [2-ß-Ribf-](n) is a short (three to five residues) oligomer of 1,2-linked ß-ribofuranose (riban), and PS is a polysaccharide chain consisting of N-acetyllactosamine, substituted with α-Fuc to form Lewis (Le)-type structures. In addition to the previously identified LacNAc, Le(y) and Le(x) components, the O-chain polysaccharide of H. pylori SS1 LPS was found to contain a novel LacNAc unit carrying a phosphoethanolamine substituent at the O-6 position of ß-GlcNAc residues.

Study of matrix additives for sensitive analysis of lipid A by matrix-assisted laser desorption ionization mass spectrometry.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been widely used for structural characterization of bacterial endotoxins (lipid A). However, the mass spectrometric behavior of the lipid A molecule is highly dependent on the matrix. Furthermore, this dependence is strongly linked to phosphorylation patterns. Using lipid A from Escherichia coli O116 as a model system, we have investigated the effects of different matrices and comatrix compounds on the analysis of lipid A. In this paper, we report a highly sensitive matrix system for lipid A analysis, which consists of 5-chloro-2-mercaptobenzothiazole matrix and EDTA ammonium salt comatrix. This matrix system enhances the sensitivity of the analysis of diphosphorylated lipid A species by more than 100-fold and in addition provides tolerance to high concentrations of sodium dodecyl sulfate (SDS) and tolerance to sodium chloride and calcium chloride at 10 muM, 100 muM, and 10 muM concentrations. The method was further evaluated for analysis of lipid A species with different phosphorylation patterns and from different bacteria, including Helicobacter pylori, Salmonella enterica serovar Riogrande, and Francisella novicida.

Characterization of polysaccharides using mass spectrometry for bacterial serotyping.

Mass spectrometry provides a rapid and reliable method for characterization of bacterial polysaccharides. Application of the in-source fragmentation technique to promote the formation of structurally relevant repeating units of heterogeneous capsular polysaccharides and O-chain polysaccharides has proven to be particularly useful for detection of non-carbohydrate functionalities and subtle differences arising across bacterial serotypes. Here we discuss application of these methods to the direct analysis of bacterial cells allowing for rapid analysis of cell surface polysaccharide antigens and providing a basis for serological typing and epidemiological surveillance studies of human and animal pathogens.

Structural characterization of the lipopolysaccharide O-antigen from atypical isolate of Vibrio anguillarum strain 1282.

Vibrio anguillarum is a Gram-negative bacterium associated with vibriosis in Atlantic cod (Gadus morhua L.). Although farmed cod in Norway is routinely vaccinated against the infection, outbreaks of V. anguillarum-associated vibriosis still occur. Here, we describe the structural characterization of the LPS O-chain polysaccharide (O-PS) from atypical isolates of V. anguillarum strain 1282 and show that it is distinct from that previously established for V. anguillarum serotype O2. The structure of the purified O-PS was shown by 1D/2D NMR ((1)H, (13)C) spectroscopy and CE-MS studies to be a high-molecular mass linear polymer of tetrasaccharide repeating units, composed of 2-acetamido-3-(N-formyl-L-alanyl)amido-2,3-dideoxy-D-glucuronamide [GlcNAc3N(Fo-L-Ala)AN], 2-acetamido-3-acetamidino-2,3-dideoxy-D-mannuronic acid (ManNAc3NAmA), 3-acetamido-3-dideoxy-D-quinovose (Qui3NAc), and 2,4-diacetamido-2,4-dideoxy-D-fucose (FucNAc4NAc). [carbohydrate structure: see text]. NMR analysis of the partial hydrolysis-derived oligosaccharides confirmed the presence of an O-acetyl group at position O-4 of GlcNAc3N(Fo-L-Ala)AN and established that the above-mentioned structure represents the biological repeating unit of the O-PS. In addition, it was demonstrated that some of 2,3-diamino-2,3-dideoxy-glucuronamide in the O-PS was present in the form of 2,3-diamino-2,3-dideoxy-glucose.

Application of an immunoaffinity-based preconcentration method for mass spectrometric analysis of the O-chain polysaccharide of Aeromonas salmonicida from in vitro- and in vivo-grown cells.

In this study, application of magnetic beads (Dynabeads) coated with Aeromonas salmonicida lipopolysaccharide-specific polyclonal antisera to MS-based characterization of bacterial lipopolysaccharides has been evaluated. The results showed that the affinity-based preconcentration strategy resulted in at least a 100-fold increase in the detection of sensitivity, affording direct capillary electrophoresis (CE)-MS analysis of A. salmonicida lipopolysaccharide O-chain polysaccharide from in vitro-cultured cells. Subsequent CE-MS analysis of in vivo-grown cells of A. salmonicida confirmed significant changes in the structure of the lipopolysaccharide O-chain polysaccharide as a result of in vivo cultivation.

Microwave-assisted sample preparation for rapid and sensitive analysis of H. pylori lipid A applicable to a single colony.

The lipid A of Gram-negative bacteria plays a major role in the pathogenesis of bacterial infections. Lipid A diversity is observed both in the number and length of fatty-acid side chains and in the presence of terminal phosphate residues and associated modifications. In this report, we describe a new sample preparation method based on microwave-assisted enzymatic digestion and detergent-free mild hydrolysis, in conjunction with a MALDI-time-of-flight (TOF)/TOF analysis, to determine the structures of lipid A from Helicobacter pylori. The total time for sample preparation and mass spectrometric analysis is within 2 h and applicable to profiling the lipid A structures from dried bacterial cells on as little as 1 microg. The reliability of the technique was further demonstrated through the analysis of the lipid A from bacterial cells of different H. pylori strains. The phosphorylation and acylation patterns of lipid A could be elucidated using material from a single colony. Furthermore, we found unusual heptaacyl lipid A species present in H. pylori mutant that have not been previously reported, although the abundance was relatively low. The present study provides the first characterization of the lipid A component from a single bacterial colony sample by mass spectrometry.