Biomedical subjects

M B Perry

Publications and source records attributed to M B Perry.

At least 37 records · Page 2Linked to original sources

PubMed2000

Lipopolysaccharide structures of Helicobacter pylori genomic strains 26695 and J99, mouse model H. pylori Sydney strain, H. pylori P466 carrying sialyl Lewis X, and H. pylori UA915 expressing Lewis B classification of H. pylori lipopolysaccharides into glycotype families.

This study describes the molecular makeup of the cell-wall lipopolysaccharides (LPSs) (O-chain polysaccharide-->core oligosaccharide-->lipid A) from five Helicobacter pylori strains: H. pylori 26695 and J99, the complete genome sequences of which have been published, the established mouse model Sydney strain (SS1), and the symptomatic strains P466 and UA915. All chemical and serological experiments were performed on the intact LPSs. H. pylori 26695 and SS1 possessed either a low-Mr semi-rough-form LPS carrying mostly a single Ley type-2 blood-group determinant in the O-chain region covalently attached to the core oligosaccharide or a high-Mr smooth-form LPS, as did strain J99, with an elongated partially fucosylated type-2 N-acetyllactosamine (polyLacNAc) O-chain polymer, terminated mainly by a Lex blood-group determinant, connected to the core oligosaccharide. In the midst of semi-rough-form LPS glycoforms, H. pylori 26695 and SS1 also expressed in the O-chain region a difucosylated antigen, alpha-L-Fucp(1-3)-alpha-L-Fucp(1-4)-beta-D-GlcpNAc, and the cancer-cell-related type-1 or type-2 linear B-blood-group antigen, alpha-D-Galp(1-3)-beta-D-Galp(1-3 or 4)-beta-D-GlcpNAc. The LPS of H. pylori strain P466 carried the cancer-associated type-2 sialyl Lex blood-group antigen, and the LPS from strain UA915 expressed a type-1 Leb blood-group unit. These findings should aid investigations that focus on identifying and characterizing genes responsible for LPS biosynthesis in genomic strains 26695 and J99, and in understanding the role of H. pylori LPS in animal model studies. The LPSs from the H. pylori strains studied to date were grouped into specific glycotype families.

Animals↗

PubMed2000

Structural identification of the lipopolysaccharide O-antigen produced by Yersinia enterocolitica serotype O:28.

The structure of the O-antigenic polysaccharide (O-PS) component of the lipopolysaccharide produced by Yersinia enterocolitica serotype O:28 has been elucidated. From chemical methods involving glycose analysis, periodate oxidation, methylation and the use of one- and two-dimensional NMR spectroscopy, the O-PS was found to be a polymer of repeating branched hexasaccharide units composed of L-rhamnose (four parts), 2-acetamido-2-deoxy-D-glucose (one part), and 2-acetamido-2-deoxy-D-galacturonic acid (one part) having the following structure:

Carbohydrate Conformation↗

PubMed2000

Structural analysis of the core region of lipopolysaccharides from Proteus mirabilis serotypes O6, O48 and O57.

The structure of lipid A core region of the lipopolysaccharides (LPS) from Proteus mirabilis serotypes O6, O57 and O48 was determined using NMR, MS and chemical analysis of the oligosaccharides, obtained by mild acid hydrolysis, alkaline deacylation, and deamination of LPS: [see text for structure]. Incomplete substitutions are indicated by bold italic type. All sugars are present in pyranose form, alpha-Hep is the residue of L-glycero-alpha-D-manno-Hep, alpha-DD-Hep is the residue of D-glycero-alpha-D-manno-Hep, L-Ara4N is 4-amino-4-deoxy-L-arabinose, Qui4NAlaAla is the residue of 4-N-(L-alanyl-L-alanyl)-4-amino-4,6-dideoxyglucose. All sugars except L-Ara4N have D-configuration. beta-GalA* is partially present in the form of amide with 1,4-diaminobutane (putrescine)-HN(CH2)4NH2 or spermidine-HN(CH2)3NH(CH2)4NH2.

Carbohydrate Conformation↗

PubMed2000

The structure of the nonreducing terminal groups in the O-specific polysaccharides from two strains of Bordetella bronchiseptica.

The structures of the polysaccharide chains of the LPS from Bordetella bronchiseptica strains 110H and Bp512 were analysed by NMR spectroscopy and mass spectrometry. The polysaccharides consist of alpha-(1-4)-linked 2,3-diacetamido-2,3-dideoxy-L-galacturonic acid repeating units. Polysaccharides from both strains have 2,3, 4-triamino-2,3,4-trideoxy-alpha-galacturonamide derivatives at their nonreducing ends, a monosaccharide identified for the first time in nature. The polymers from the two strains differ in the nature of the acylation of the amino groups of this monosaccharide. In the strain 110H, the residue is formylated at positions 3 and 4, and has N-formyl-L-alanyl or L-alanyl substituents at N-2. In the strain Bp512, the amino group at position 2 is acetylated, at position 3 it is formylated, and the amino group at position 4 bears a 2-methoxypropionyl substituent. The distribution of the acyl groups was determined from long range 1H-13C correlation (HMBC) NMR spectra. Measurement of the spectra under different pH conditions showed that carboxyl groups of the inner uronic acid residues of the polymeric chain are free, and that carboxyl groups of the terminal residues are amidated. These conclusions were confirmed by the results of mass spectrometric analysis.

Bordetella bronchiseptica↗

PubMed2000

Expression of histo-blood group antigens by lipopolysaccharides of Helicobacter pylori strains from asian hosts: the propensity to express type 1 blood-group antigens.

Past studies have shown that the cell surface lipopolysaccharides (LPSs) of the ubiquitous human gastric pathogen Helicobacter pylori (a type 1 carcinogen) isolated from people residing in Europe and North America express predominantly type 2 Lewis x (Le(x)) and Le(y) epitopes and, infrequently, type 1 Le(a), Le(b), and Le(d) antigens. This production of Lewis blood-group structures by H. pylori LPSs, similar to those found in the surfaces of human gastric cells, allows the bacterium to mimic its human niche. In this study, LPSs of H.pylori strains extracted from patients living in China, Japan, and Singapore were chemically and serologically analyzed. When compared with Western H.pylori LPSs, these Asian strains showed a stronger tendency to produce type 1 blood groups. Of particular interest, and novel observations in H.pylori, the O-chain regions of strains F-58C and R-58A carried type 1 Le(a) without the presence of type 2 Le(x), strains R-7A and H607 were shown to have the capability of producing the type 1 blood group A antigen, and strains CA2, H507, and H428 expressed simultaneously the difucosyl isomeric antigens, type 1 Le(b) and type 2 Le(y). The apparent proclivity for the production of type 1 histo-blood group antigens in Asian H.pylori LPSs, as compared with Western strains, may be an adaptive evolutionary effect in that differences in the gastric cell surfaces of the respective hosts might be significantly dissimilar to select for the formation of different LPS structures on the resident H.pylori strain.

ABO Blood-Group System↗

PubMed2000

Mutation of the lipopolysaccharide core glycosyltransferase encoded by waaG destabilizes the outer membrane of Escherichia coli by interfering with core phosphorylation.

In Escherichia coli, phosphoryl substituents in the lipopolysaccharide core region are essential for outer membrane stability. Mutation of the core glucosyltransferase encoded by waaG (formerly rfaG) resulted in lipopolysaccharide truncated immediately after the inner core heptose residues, which serve as the sites for phosphorylation. Surprisingly, mutation of waaG also destabilized the outer membrane. Structural analyses of waaG mutant lipopolysaccharide showed that the cause for this phenotype was a decrease in core phosphorylation, an unexpected side effect of the waaG mutation.

Acylation↗

PubMed2000

Prevalence of O1/K1- and O2/K3-reactive Actinobacillus suis in healthy and diseased swine.

A cell surface antigen-typing system was devised for the swine pathogen Actinobacillus suis and used to examine the prevalence of different lipopolysaccharide (O) types in healthy and diseased pigs. The strains examined in this study were isolated from a variety of locations in Canada and from Kansas. Lipopolysaccharide preparations of 151 isolates of A. suis were characterized by immunoblotting using polyclonal antisera generated to strains SO4 (O1/K1), H89-1173 (O2/K3), and VSB 3714, a rough strain. Approximately 54% (62 of 114) of A. suis isolates from diseased pigs, all (11 of 11) isolates from healthy pigs, and all (4 of 4) reference strains reacted with O1/K1 antiserum. More than 80% (18 of 22) of A. suis strains used for bacterin production and approximately 41% (47 of 114) of isolates from diseased pigs bound O2/K3 antiserum. One isolate appeared to be rough, and five were untypeable. O1/K1- and O2/K3-reactive strains were equally prevalent in Kansas, whereas O2/K3-reactive strains were more common in Québec and western Canada and O1/K1 strains were most common in Ontario. The fact that virtually all of the strains submitted for bacterin production were O2/K3-reactive strains is consistent with the notion that these strains may be more virulent than O1/K1 strains; alternatively, this may reflect geographic or other biases. In addition, we observed cross-reactivity between A. suis cell surface antigens and swine antisera to several other important pathogens. This finding may explain why previous attempts to develop a simple serodiagnostic test for A. suis have been unsuccessful.

Actinobacillus↗

PubMed2000

Oral immunization of mice with a glycoconjugate vaccine containing the O157 antigen of Escherichia coli O157:H7 admixed with cholera toxin fails to elicit protection against subsequent colonization by the pathogen.

It has been postulated that a humoral immune response directed against the O157 antigen of Escherichia coli O157:H7, and expressed in the intestine, might afford protection from colonization and consequent infection by this enteric pathogen. The present study was conducted to determine whether such an immune response can be experimentally generated in mice. To this end, mice were orally immunized with a glycoconjugate vaccine consisting of horse serum albumin and the O157 polysaccharide admixed with the mucosal adjuvant, cholera toxin. Mice consistently developed robust local and systemic immune responses to the cholera toxin adjuvant, but were far from uniformly reactive to the test vaccine. Moreover, vaccinated mice were as susceptible to transient intestinal colonization following challenge with an isolate of E. coli O157:H7 as unvaccinated control mice. These results indicate that this vaccination approach is unlikely to be straightforward in target bovine or human hosts.

Animals↗

PubMed1999

The activity of a putative polyisoprenol-linked sugar translocase (Wzx) involved in Escherichia coli O antigen assembly is independent of the chemical structure of the O repeat.

During O antigen lipopolysaccharide (LPS) synthesis in bacteria, transmembrane migration of undecaprenylpyrophosphate (Und-P-P)-bound O antigen subunits occurs before their polymerization and ligation to the rest of the LPS molecule. Despite the general nature of the translocation process, putative O-antigen translocases display a low level of amino acid sequence similarity. In this work, we investigated whether complete O antigen subunits are required for translocation. We demonstrate that a single sugar, GlcNAc, can be incorporated to LPS of Escherichia coli K-12. This incorporation required the functions of two O antigen synthesis genes, wecA (UDP-GlcNAc:Und-P GlcNAc-1-P transferase) and wzx (O-antigen translocase). Complementation experiments with putative O-antigen translocases from E. coli O7 and Salmonella enterica indicated that translocation of O antigen subunits is independent of the chemical structure of the saccharide moiety. Furthermore, complementation with putative translocases involved in synthesis of exopolysaccharides demonstrated that these proteins could not participate in O antigen assembly. Our data indicate that recognition of a complete Und-P-P-bound O antigen subunit is not required for translocation and suggest a model for O antigen synthesis involving recognition of Und-P-P-linked sugars by a putative complex made of Wzx translocase and other proteins involved in the processing of O antigen.

Agglutination↗

PubMed1999

Structural characterization of the antigenic O-chain of the lipopolysaccharide of Escherichia coli serotype O65.

The antigenic O-polysaccharide moiety of the lipopolysaccharide produced by Escherichia coli serotype O65 was investigated by composition, methylation, base hydrolysis, periodate oxidation, mass spectrometric methods, and by 1D and 2D NMR spectroscopy. The O-polysaccharide had [alpha]D + 108 degrees (water) and is a high-molecular-weight unbranched linear polymer of repeating pentasaccharide units composed of 1:1:1:1:1 D-galacturonic acid (D-GalA), D-galacturonamide (D-GalANH2), 2-acetamido-2-deoxy-D-glucose (D-GlcNAc), 2-acetamido-2-deoxy-D-galactose (D-GalNAc), and 3-acetamido-3,6-dideoxy-D-glucose (D-Qui3NAc), and has the following structure: [formula: see text]

Carbohydrate Conformation↗

PubMed1999

Phase variation in Helicobacter pylori lipopolysaccharide due to changes in the lengths of poly(C) tracts in alpha3-fucosyltransferase genes.

The lipopolysaccharide (LPS) of Helicobacter pylori expresses the Lewis x (Lex) and/or Ley antigen. We have shown previously that H. pylori LPS displays phase variation whereby an Lex-positive strain yields variants with different LPS serotypes, for example, Lex plus Ley or nonfucosylated polylactosamine. H. pylori has two alpha3-fucosyltransferase genes that both contain poly(C) tracts. We now demonstrate that these tracts can shorten or lengthen randomly, which results in reversible frameshifting and inactivation of the gene products. We provide genetic and serological evidence that this mechanism causes H. pylori LPS phase variation and demonstrate that the on or off status of alpha3-fucosyltransferase genes determines the LPS serotypes of phase variants and clinical isolates. The role of the alpha3-fucosyltransferase gene products in determining the LPS serotype was confirmed by structural-chemical analysis of alpha3-fucosyltransferase knockout mutants. The data also show that the two alpha3-fucosyltransferase genes code for enzymes with different fine specificities, and we propose the names futA and futB to designate the orthologs of the H. pylori 26695 alpha3-fucosyltransferase genes HP0379 and HP0651, respectively. The data also show that the alpha3-fucosylation precedes alpha2-fucosylation [corrected], an order of events opposite to that which prevails in mammals. Finally, the data provide an understanding at the molecular level of the mechanisms underlying LPS diversity in H. pylori, which may play an important role in adaptation to the host.

Fucosyltransferases↗

PubMed1999

Parenteral immunization with a glycoconjugate vaccine containing the O157 antigen of Escherichia coli O157:H7 elicits a systemic humoral immune response in mice, but fails to prevent colonization by the pathogen.

The results of the present study show that whereas both BALB/c and C57BL/6 mice parenterally inoculated with a horse serum albumin-Escherichia coli O157 antigen conjugate vaccine develop systemic, specific antibodies to the carrier protein, only the former mice routinely develop antibodies to the carbohydrate O157 moiety. However, little convincing evidence was found to show that these antibodies transuded into the intestinal tract either naturally or in response to an oral inoculum of the pathogen. Moreover, this vaccination procedure failed to protect mice against intestinal colonization following oral challenge with the pathogen. Thus, the results of this study suggest that parenteral vaccination might be an unsuitable strategy for combatting E. coli O157:H7 organisms located in the gut.

Animals↗

PubMed1999

Salmonella landau as a live vaccine against Escherichia coli O157:H7 investigated in a mouse model of intestinal colonization.

The present study was performed to assess the potential of a humoral mucosal immune response directed against the O157 antigen of Escherichia coli O157:H7 to prevent intestinal colonization by the pathogen. To this end, mice were gavaged with inocula of Salmonella landau, a Salmonella strain that naturally expresses the O157 antigen. Salmonella landau was avirulent for mice. Despite this, mice exposed to S. landau developed high titres of serum and coproantibodies against the O157 antigen. These mice, compared with controls, demonstrated some ability to resist transient intestinal colonization by an oral inoculum of an isolate of E. coli O157:H7. These findings suggest that a local immune response directed against the O157 antigen might increase host resistance to this pathogen.

Administration, Oral↗

PubMed1999

Imunosuppression by a corticosteroid fails to exacerbate Helicobacter pylori infection in a mouse model of gastric colonization.

Helicobacter pylori can colonize the human stomach for prolonged periods of time, and this colonization uniformly leads to the development of chronic active gastritis. In a small percentage of individuals, gastric pathology progresses to peptic ulceration or more rarely certain gastric cancers. In addition to non-specific inflammation, specific systemic and local immunity develops in response to gastric colonization by this pathogen. However, these responses combined appear inadequate for eliminating H. pylori from the gastric mucosa. This is also the case in a mouse model of gastric colonization by H. pylori. In the present study, we attempted to determine whether the mammalian host response to infection with H. pylori exerts any overt antibacterial effects. To this end we examined H. pylori colonization in normal mice, and mice immunosuppressed by treatment with a corticosteroid. Despite obvious suppression of the immune response in the latter mice, H. pylori burdens remained similar in both groups after three months of colonization. This suggests that the murine host response, at least, exerts little obvious protection against H. pylori colonization.

Animals↗

PubMed1998

Involvement of waaY, waaQ, and waaP in the modification of Escherichia coli lipopolysaccharide and their role in the formation of a stable outer membrane.

The waaY, waaQ, and waaP genes are located in the central operon of the waa (formerly rfa) locus on the chromosome of Escherichia coli. This locus contains genes whose products are involved in the assembly of the core region of the lipopolysaccharide molecule. In the R1 core prototype strain, E. coli F470, there are nine genes in this operon, and all but waaY, waaQ, and waaP have been assigned function. In this study, the waaY, waaQ, and waaP genes were independently mutated by insertion of a non-polar antibiotic resistance cassette, and the structures of the resulting mutant core oligosaccharides were determined by chemical analyses and phosphorus-nuclear magnetic resonance spectroscopy. All three of these mutations were shown to affect the modification of the heptose region of the core, a region whose structure is critical to outer membrane stability. Mutation of waaY resulted in a core oligosaccharide devoid of phosphate on HepII. Mutation of waaQ resulted in loss of the branch HepIII residue on HepII and impeded the activity of WaaY. Mutation of waaP resulted in loss of phosphoryl substituents on HepI and obviated WaaQ and WaaY activity. Only mutation of waaP resulted in hypersensitivity to novobiocin and sodium dodecyl sulfate, a characteristic of deep-rough mutations.

Base Sequence↗

PubMed1998

Simultaneous expression of type 1 and type 2 Lewis blood group antigens by Helicobacter pylori lipopolysaccharides. Molecular mimicry between h. pylori lipopolysaccharides and human gastric epithelial cell surface glycoforms.

Previous structural investigations performed on the lipopolysaccharides (LPSs) from the human gastric pathogen Helicobacter pylori have revealed that these cell surface glycan molecules express type 2 partially fucosylated, glucosylated, or galactosylated N-acetyllactosamine O antigen chains (O-chains) of various lengths, which may or may not be terminated at the nonreducing end by Lewis X (Lex) and/or Ley blood group epitopes in mimicry of human cell surface glycoconjugates and glycolipids. Subsequently, serological experiments with commercially available Lewis-specific monoclonal antibodies also have recognized the presence of Lex and Ley blood group antigens in H. pylori but, in addition, have indicated the presence of type 1 chain Lea, Leb, and Led (H-type 1) blood group epitopes in some H. pylori strains. To confirm their presence, structural studies and additional serological experiments were undertaken on H. pylori strains suspected of carrying type 1 chain epitopes. These investigations revealed that the O-chain region of H. pylori strain UA948 carried both Lea (type 1) and Lex (type 2) blood group determinants. The O-chain from H. pylori UA955 LPS expressed the terminal Lewis disaccharide (type 1 chain) and Lex and Ley antigens (type 2). The O-chain of H. pylori J223 LPS carried the type 1 chain precursor Lec, the H-1 epitope (Led, type 1 chain) and an elongated nonfucosylated type 2 N-acetyllactosamine chain (i antigen). Thus, O-chains from H. pylori LPSs can also express fucosylated type 1 sequences, and the LPS from a single H. pylori strain may carry O-chains with type 1 and 2 Lewis blood groups simultaneously. That monoclonal antibodies putatively specific for the Leb determinant can detect glycan substructures (Le disaccharide, Lec, and Led) of Leb indicates their nonspecificity. The expression of both type 1 and 2 Lewis antigens by H. pylori LPSs mimics the cell surface glycomolecules present in both the gastric superficial (which expresses mainly type 1 determinants) and the superficial and glandular epithelium regions (both of which express predominantly type 2 determinants). Therefore, each H. pylori strain may have a different niche within the gastric mucosa, and each individual LPS blood group antigen may have a dissimilar role in H. pylori adaptation.

Carbohydrate Sequence↗

PubMed1998

Structural analysis of the phase-variable lipooligosaccharide from Haemophilus somnus strain 738.

The structure of the phase variable lipooligosaccharide (LOS) from Haemophilus somnus strain 738 was elucidated. The LOS was subjected to a variety of degradative procedures. The structures of the purified products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structures for the two major components were determined on the basis of the combined data from these experiments. [structure in text]. In the structures Kdo is 3-deoxy-D-manno-octulosonic acid, PEtn is phosphoethanolamine, PCho is phosphocholine, Hep is L-glycero-D-manno-heptose, and the remaining glucose units have the D configuration. The elucidation of these structures has increased our understanding of the relationship between the phase-variable LOS and the pathogenic potential of this organism.

Haemophilus↗

PubMed1998

The assembly system for the lipopolysaccharide R2 core-type of Escherichia coli is a hybrid of those found in Escherichia coli K-12 and Salmonella enterica. Structure and function of the R2 WaaK and WaaL homologs.

In Escherichia coli F632, the 14-kilobase pair chromosomal region located between waaC (formerly rfaC) and waaA (kdtA) contains genes encoding enzymes required for the synthesis of the type R2 core oligosaccharide portion of lipopolysaccharide. Ten of the 13 open reading frames encode predicted products sharing greater than 90% total similarity with homologs in E. coli K-12. However, the products of waaK (rfaK) and waaL (rfaL) each resemble homologs in Salmonella enterica serovar Typhimurium but share little similarity with E. coli K-12. The F632 WaaK and WaaL proteins therefore define differences between the type R2 and K-12 outer core oligosaccharides of E. coli lipopolysaccharides. Based on the chemical structure of the core oligosaccharide of an E. coli F632 waaK::aacC1 mutant and in vitro glycosyltransferase analyses, waaK encodes UDP-N-acetylglucosamine:(glucose) lipopolysaccharide alpha1, 2-N-acetylglucosaminyltransferase. The WaaK enzyme adds a terminal GlcNAc side branch substituent that is crucial for the recognition of core oligosaccharide acceptor by the O-polysaccharide ligase, WaaL. Results of complementation analyses of E. coli K-12 and F632 waaL mutants suggest that structural differences between the WaaL proteins play a role in recognition of, and interaction with, terminal lipopolysaccharide core moieties.

Amino Acid Sequence↗