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PubMed1985

Chemical characterization of capsular polysaccharide from Cryptococcus neoformans serotype A-D.

During a study of serotyping of Cryptococcus neoformans, we found that the type strain of C. neoformans (CBS 132) was serotype A-D. This strain agglutinated with both factor 7 serum (specific for serotype A) and factor 8 serum (specific for serotype D) in our serotyping system. Therefore, we investigated the chemical structure of the antigenic capsular polysaccharide of this strain. The soluble capsular polysaccharide was obtained from the culture supernatant fluid by precipitation with ethanol. Column chromatography of the polysaccharide on DEAE-cellulose yielded three fractions (F-1 to F-3). The major antigenic activity was found in the F-3 fraction. The results obtained by methylation analysis, controlled Smith degradation-methylation analysis, partial acid hydrolysis, and other structural studies of F-3 polysaccharide indicated that the polysaccharide contains mannose, xylose, and glucuronic acid at a ratio of 7:2:2, and has a backbone of alpha (1-3)-linked D-mannopyranoside residues with a single branch of beta (1-2)-xylose and glucuronic acid. The ratio of mannose residues with or without a branch in the F-3 polysaccharide was 4:3 and its molecular weight calculated from the average of the degree of polymerization was 46,500 daltons. These results indicate that the chemical structure of the capsular polysaccharide of serotype A-D is very similar to those from serotypes A and D, suggesting that small differences in the molar ratio and pattern of linkage of monosaccharides in the branch of the polysaccharides of the three serotypes may be responsible for their different specificities.

Antigens, Bacterial↗
PubMed1993

Structural variation in the O-specific polysaccharides of Klebsiella pneumoniae serotype O1 and O8 lipopolysaccharide: evidence for clonal diversity in rfb genes.

The O-polysaccharide fraction of the lipopolysaccharide from Klebsiella pneumoniae serotype O8 was found to comprise two galactose-containing homopolymers. Structural analysis, using chemical and high-field nuclear magnetic resonance (NMR) techniques, established that the K. pneumoniae O8 polysaccharides are composed of the linear, disaccharide repeating units [formula: see text] K. pneumoniae O8 mutant RFK-1 was isolated by resistance to phage KO1-2; strain RFK-1 expressed only D-galactan I-OAc. The 1H- and 13C-NMR resonances from this O-polysaccharide indicate that all of the O-acetyl groups within the K. pneumoniae O8 polysaccharide are carried on D-galactan I and O-acetylation occurs only on the beta-D-galactofuranose residues; 60% of the available beta-D-galactofuranose residues are non-acetylated. The O-acetylation of the remaining residues is equally distributed between the O-2 and O-6 positions. The carbohydrate backbone structures in the O8 polysaccharide are identical to D-galactan I and II expressed by K. pneumoniae O1, accounting for the antigenic cross-reaction between strains belonging to serotypes O1 and O8. However, the O1 polysaccharides are not acetylated and the O-acetyl groups present in the K. pneumoniae serotype O8 polysaccharides provide a structural basis for their recognition as distinct serotypes. The rfb (O-polysaccharide biosynthesis) gene cluster of K. pneumoniae serotype O1 determines the synthesis of D-galactan I. rfbKpO1-specific gene probes were used to examine conservation in the rfb gene clusters of other K. pneumoniae serotypes which produce D-galactan I. Six O1 strains were examined and all showed hybridization with rfbKpO1 probes under conditions of high stringency. Three serotype O2 strains produce D-galactan I and these strains also contained DNA sequences recognized by rfbKpO1 probes under high stringency. The physical maps of these homologous rfb chromosomal regions showed some polymorphism. Surprisingly, the rfbKpO8 region from K. pneumoniae serotype O8 was only recognized by rfbKpO1 probes under low-stringency hybridization conditions, providing evidence for two substantially different clonal groups of rfb genes from K. pneumoniae strains with structurally related O-antigens.

Acetylation↗
PubMed1993

Phospholipid substitution of capsular polysaccharides and mechanisms of capsule formation in Neisseria meningitidis.

Within the capsule gene complex (cps) of Neisseria meningitidis two functional regions B and C are involved in surface translocation of the cytoplasmically synthesized capsular polysaccharide, which is a homopolymer of alpha-2,8 polyneuraminic acid. The region-C gene products share characteristics with transporter proteins of the ABC (ATP-binding cassette) superfamily of active transporters. For analysis of the role of region B in surface translocation of the capsular polysaccharide we purified the polysaccharides of region B- and region C-defective Escherichia coli clones by affinity chromatography. The molecular weights of the polysaccharides were determined by gel filtration and the polysaccharides were analysed for phospholipid substitution by polyacrylamide gel electrophoresis and immunoblotting. The results indicate that the full-size capsular polysaccharide with a phospholipid anchor is synthesized intracellularly and that lipid modification is a strong requirement for translocation of the polysaccharide to the cell surface. Proteins encoded by region B are involved in phospholipid substitution of the capsular polysaccharide. Nucleotide sequence analysis of region B revealed two open reading frames, which encode proteins with molecular masses of 45.1 and 48.7 kDa.

Amino Acid Sequence↗
PubMed2006

A novel polar surface polysaccharide from Rhizobium leguminosarum binds host plant lectin.

Rhizobium bacteria produce different surface polysaccharides which are either secreted in the growth medium or contribute to a capsule surrounding the cell. Here, we describe isolation and partial characterization of a novel high molecular weight surface polysaccharide from a strain of Rhizobium leguminosarum that nodulates Pisum sativum (pea) and Vicia sativa (vetch) roots. Carbohydrate analysis showed that the polysaccharide consists for 95% of mannose and glucose, with minor amounts of galactose and rhamnose. Lectin precipitation analysis revealed high binding affinity of pea and vetch lectin for this polysaccharide, in contrast to the other known capsular and extracellular polysaccharides of this strain. Expression of the polysaccharide was independent of the presence of a Sym plasmid or the nod gene inducer naringenin. Incubation of R. leguminosarum with labelled pea lectin showed that this polysaccharide is exclusively localized on one of the poles of the bacterial cell. Vetch roots incubated with rhizobia and labelled pea lectin revealed that this bacterial pole is involved in attachment to the root surface. A mutant strain deficient in the production of this polysaccharide was impaired in attachment and root hair infection under slightly acidic conditions, in contrast to the situation at slightly alkaline conditions. Our data are consistent with the hypothesis that rhizobia can use (at least) two mechanisms for docking at the root surface, with use of a lectin-glycan mechanism under slightly acidic conditions.

Carbohydrates↗
PubMed1993

Modification of experimental Porphyromonas gingivalis murine infection by immunization with a polysaccharide-protein conjugate.

To better understand the role of the capsular polysaccharide in the virulence of Porphyromonas gingivalis, the effect of immunization with a polysaccharide-protein conjugate on experimental murine infection was evaluated. The conjugate was prepared using polysaccharide isolated from P. gingivalis strain ATCC 53977 and bovine serum albumin. One group of 22 mice was immunized by intraperitoneal injection with the conjugate and a control group of 25 mice was similarly immunized with bovine serum albumin. Serum antibody reactive to the polysaccharide, as determined by enzyme-linked immunosorbent assay, was elevated in the group of mice immunized with the polysaccharide-protein conjugate but not in the mice immunized with bovine serum albumin. Both groups of mice were challenged with P. gingivalis strain ATCC 53977 (10(10) cells) administered subcutaneously on the dorsal surface. Following challenge, the mice immunized with the polysaccharide-protein conjugate appeared healthier and demonstrated less weight loss than did the control group of mice. Ulcerative lesions at secondary locations were smaller in mice immunized with the polysaccharide-protein conjugate. Thus, immunization of mice with a conjugate containing P. gingivalis polysaccharide could reduce the severity of but not prevent an invasive infection with P. gingivalis.

Animals↗
PubMed1995

Role of prostaglandin in the formation of osteoclasts induced by capsular-like polysaccharide antigen of Actinobacillus actinomycetemcomitans strain Y4.

We found no reports that capsular-like polysaccharide antigen purified from Actinobacillus actinomycetemcomitans either induces osteoclastic bone resorption in mouse organ cultures or promotes osteoclast formation in mouse marrow cultures. In contrast, capsular-like polysaccharide antigen purified from A. actinomycetemcomitans strain Y4 induced bone resorption in mouse organ culture. To examine the mechanism of bone resorption induced by A. actinomycetemcomitans, mouse bone marrow cells were cultured with A. actinomycetemcomitans strain Y4 capsular-like polysaccharide antigen. A. actinomycetemcomitans strain Y4 capsular-like polysaccharide antigen stimulated osteoclast-like cell formation in mouse bone marrow cultures. However, the polysaccharide of A. actinomycetemcomitans lipopolysaccharide did not induce the formation of osteoclast-like cells. Indomethacin inhibited osteoclast-like cell formation mediated by A. actinomycetemcomitans strain Y4 capsular-like polysaccharide antigen in a dose-dependent manner. There was a good correlation between the number of osteoclast-like cells formed in the marrow culture and the amount of prostaglandin E2 released into the culture media. When mouse bone marrow cells were cultured with prostaglandin E2 during the culture periods, many osteoclast-like cells were formed. These results indicate that prostaglandin E2 is involved in the mechanism of the formation of osteoclast-like cells mediated by A. actinomycetemcomitans strain Y4 capsular-like polysaccharide antigen. A. actinomycetemcomitans strain Y4 capsular-like polysaccharide antigen may play an important role in inflammatory bone resorption by promoting osteoclast formation in periodontal disease.

Acid Phosphatase↗
PubMed1986

Polysaccharide from coccoliths (CaCO3 biomineral). Influence on crystallization of calcium oxalate monohydrate.

A polysaccharide associated with coccoliths of the marine alga Emiliania huxleyi (coccoliths are elaborately shaped calcite biominerals) was isolated and its influence on the crystallization of calcium oxalate monohydrate crystals was studied. Crystallization was monitored in a carefully controlled system by measuring the incorporation of 45Ca tracer from a supersaturated solution into seed crystals of calcium oxalate monohydrate in the absence and in the presence of polysaccharide. The method allowed differentiation between effects on solubility, growth and agglomeration of crystals. At the very low concentrations used in this study, the polysaccharide had no significant effect on the solubility product; it strongly inhibited the growth and strongly stimulated the agglomeration of the crystals. Thus, the two processes of growth and agglomeration, being both crystal-surface-related processes, may react in opposite directions upon surface adhesion of the additive. This finding opens new insights on how a mineralization process may be controlled. The inhibitory effect on growth is shown to proceed through a monolayer type of adsorption of the polysaccharide onto the crystal surface. The portion of the polysaccharide used for the stimulatory effect on agglomeration shows a different type of adsorption, whereby less crystal surface is covered per molecule of polysaccharide. This strongly suggests, that the mechanism whereby agglomeration is stimulated operates through 'viscous binding', with the polysaccharide bridging the gap between two crystal surfaces. In the discussion these findings are related to some possible biological functions of the polysaccharide.

Calcification, Physiologic↗
PubMed1976

Antigenicity of type-specific pneumococcal polysaccharides in rats.

Hemagglutinating antibody responses of Lewis-Wistar and Sprague-Dawley rats to graded doses of type-specific pneumococcal polysaccharide were measured. Rats given a small dose (0.2 to 50 mug)of type 1 or 8 polysaccharide intraperitoneally developed type-specific hemagglutinating antibody. Rats given larger doses of polysaccharide (greater than or equal to200 mug) did not develop detectable hemagglutinating antibody, and they were unresponsive to a subsequence injection of a small (and normally antigenic) dose of polysaccharide. There was prolonged antigenemia in rats injected with a large dose of polysaccharide. There was prolonged antigenemia in rats injected with a large dose of polysaccharide, and the kinetics of antigen clearance in these animals resembled that reported for mice with polysaccharide immunological paralysis. These results indicate that a phenomenon resembling immunological paralysis with type-specific pneumococcal polysaccharides can be produced in rats.

Animals↗
PubMed1977

Immune response to Cryptococcus neoformans soluble polysaccharide: immunological unresponsiveness.

Mice injected with 100 to 800 microgram of Cryptococcus neoformans soluble polysaccharide showed a reduced ability to produce antibody after a challenge immunization with polysaccharide emulsified in Freund incomplete adjuvant. These animals were considered immunologically unresponsive. Animals given an initial injection of 25 or 50 microgram of polysaccharide responded to a challenge immunization in the same manner as control animals. Reversion of unresponsive mice to antibody production without further antigenic stimulation did not occur during a 12-week experimental period. These animals exhibited a partial response to challenge immunization 8 weeks after induction of unresponsiveness, and they were fully responsive to challenge immunization at 12 weeks. Animals given a single dose of 0.1, 0.4, or 1.6 microgram of polysaccharide produced a marked anamnestic response after challenge immunization. Repeated injections of subimmunogenic doses of polysaccharide did not produce a marked anamnestic response and would induce unresponsiveness only when the cumulative dose reached 100 to 400 microgram of polysaccharide, suggesting that injected cryptococcal polysaccharide might be sequestered in some manner until an amount of antigen sufficient for induction of unresponsiveness is accumulated. This possibility was confirmed by immunofluorescence studies that revealed a long-term deposition of polysaccharide in the tubular epithelial cells of the kidney.

Animals↗
PubMed1980

Antigenicity and protective effects of type 3 pneumococcal polysaccharide in rats.

The response to type 3 pneumococcal polysaccharide vaccination, the protective effect of type 3 pneumococcal polysaccharide vaccination, and the ability of hemagglutinating antibody to type 3 pneumococcal polysaccharide to cross the blood-brain barrier were studied in rats. Hemagglutinating antibody response to vaccination with type 3 pneumococcal polysaccharide was found to be dependent on the dose and route of inoculation. Intraperitoneal vaccination with type 3 pneumococcal polysaccharide protected the rats from subsequent intraperitoneal challenge with type 3 Streptococcus pneumoniae but had no effect after intracisternal challenge with type 3 S. pneumoniae. In the presence or absence of sterile meningeal inflammation, hemagglutinating antibody could not be found in cerebrospinal fluid of animals vaccinated intraperitoneally with type 3 pneumococcal polysaccharide. These results suggest that type 3 pneumococcal polysaccharide is antigenic in rats and intraperitoneal vaccination with type 3 pneumococcal polysaccharide does not protect against experimental meningitis because antibodies do not cross the blood-brain barrier.

Agglutinins↗
PubMed1983

Isolation, chemical composition, and molecular size of extracellular type II and type Ia polysaccharides of group B streptococci.

Polysaccharides carrying the type II- and type Ia-specific determinants of Lancefield group B streptococci were isolated and purified by anion-exchange chromatography and gel filtration from the supernatant culture medium after growth of strain 18RS21/67/1 (type II) and strain DS/1204/78 (type Ia), respectively. The average molecular weights of these polysaccharides were 97,000 (type II) and 94,000 (type Ia), as determined by reducing end group analyses. These molecular weights were in reasonably good agreement with molecular weights determined by gel filtration at high ionic strength on calibrated columns. The polysaccharides did not cross-react with antisera specific for the other type-specific determinants or with group B-specific antisera. Their content of galactose, glucose, glucosamine, and neuraminic acid (the last two calculated as N-acetyl derivatives) accounted for over 96% of their dry weight. The two polysaccharides differed from each other (and from type III polysaccharide) in their relative content of these monosaccharides. The molar ratios of galactose, glucose, and neuraminic acid to glucosamine were 3.3:2.3:1.35:1.0 for the type II polysaccharide and 2.0:0.8:1.4:1.0 for the type Ia polysaccharides. The results obtained indicate that these extracellular type II and Ia polysaccharides contain larger amounts of neuraminic acid than can be accounted for by previously proposed structures of their repeating units.

Antigens, Bacterial↗
PubMed1988

Characterization of Brucella polysaccharide B.

Polysaccharide B was extracted from Brucella melitensis 16M and from a rough strain of Brucella abortus 45/20 by autoclaving or trichloroacetic acid extraction of whole cells and by a new method involving mild leaching of cells. The material obtained by either of the established procedures was contaminated by O polysaccharide. The new leaching protocol eliminated this impurity and provided a pure glucan, which was regarded as polysaccharide B. This polysaccharide was found by high-performance liquid chromatography separations, chemical composition, methylation, and two-dimensional homo- and heteronuclear magnetic resonance experiments to be a family of nonreducing cyclic 1,2-linked polymers of beta-D-glucopyranosyl residues. The degree of polymerization varied between 17 and 24. Polysaccharide B was essentially identical to cyclic D-glucans produced by Rhizobia, Agrobacteria, and other bacterial species. Pure polysaccharide B did not precipitate with Brucella anti-A or anti-M serum and did not inhibit the serological reaction of Brucella A or M antigen with either bovine or murine monoclonal Brucella anti-A or anti-M serum. Previously described serological reactions of polysaccharide B preparations with Brucella anti-A and anti-M sera are related in this study to the presence in crude extracts of contaminants with the antigenic properties of Brucella lipopolysaccharide O polysaccharides.

Antigens, Bacterial↗
PubMed1993

Stimulation of protective antibodies against type Ia and Ib group B streptococci by a type Ia polysaccharide-tetanus toxoid conjugate vaccine.

Antisera elicited by type Ia group B streptococci (GBS) contain antibodies that react with both type Ia and type Ib strains. Previous studies suggested that antibodies elicited by type Ia organisms recognized a carbohydrate antigen or epitope common to Ia and Ib strains. We now report the synthesis and immunogenicity testing of a type Ia polysaccharide-tetanus toxoid (Ia-TT) conjugate vaccine. Ia-TT elicited type Ia polysaccharide-specific immunoglobulin G antibodies in all three of the rabbits inoculated. In competitive enzyme-linked immunosorbent assay, these antibodies reacted with high affinity to type Ia polysaccharide and with lower affinity to the structurally related GBS type Ib polysaccharide. Despite the lower binding affinity of the Ia-TT-induced antibodies for the type Ib polysaccharide, Ia-TT antiserum opsonized not only type Ia GBS but also type Ib GBS for killing by human blood leukocytes. Ia-TT antiserum was also evaluated in a mouse model designed to test the efficacy of maternal antibodies in protecting neonates against GBS infection. Pups born to dams that had received Ia-TT antiserum were protected against lethal challenge with either type Ia or Ib GBS. These studies using a polysaccharide-protein conjugate as an immunogen support the view that the carbohydrate immunodeterminant recognized on Ib strains by Ia antisera is a common epitope contained within the structurally related Ia and Ib capsular polysaccharides. Although antibodies elicited by Ia-TT had protective activity against both Ia and Ib strains, these antibodies reacted with lower affinity to Ib than to Ia polysaccharide.

Animals↗
PubMed1994

Neonatal mouse protection against infection with multiple group B streptococcal (GBS) serotypes by maternal immunization with a tetravalent GBS polysaccharide-tetanus toxoid conjugate vaccine.

Most cases of neonatal sepsis and meningitis caused by group B streptococci (GBS) are attributable to one of four major capsular serotypes: Ia, Ib, II, or III. Because resistance to infection with GBS has been correlated with the presence of serum antibodies to the type-specific capsular polysaccharides in both experimental animals and human neonates, efforts have been made to elicit protective immunity with GBS capsular polysaccharide vaccines. However, the GBS capsular polysaccharides alone are not highly immunogenic in either animals or human volunteers. Therefore, we and other investigators have attempted to enhance immunogenicity by coupling individual capsular polysaccharides to a carrier protein. Here we report the synthesis and immunogenicity in rabbits of a GBS type Ib polysaccharide-tetanus toxoid vaccine prepared by the direct, covalent attachment of tetanus toxoid to a selected number of sialic acid residues on the type-specific polysaccharide. In addition, the Ib polysaccharide-tetanus toxoid conjugate vaccine was combined with similar tetanus toxoid conjugates of GBS type Ia, II, and III polysaccharides to form a tetravalent GBS conjugate vaccine. Protective efficacy of the GBS tetravalent conjugate vaccine was demonstrated in a mouse maternal immunization-neonatal challenge model of GBS infection. The results support testing in human subjects of a multivalent GBS conjugate vaccine of this design, with the eventual goal of protecting newborns against GBS infection.

Animals↗
PubMed1995

Adherence of Candida albicans to a cell surface polysaccharide receptor on Streptococcus gordonii.

Candida albicans ATCC 10261 and CA2 bound to cells of the oral bacteria Streptococcus gordonii, Streptococcus oralis, and Streptococcus sanguis when these bacteria were immobilized onto microtiter plate wells, but they did not bind to cells of Streptococcus mutans or Streptococcus salivarius. Cell wall polysaccharide was extracted with alkali from S. gordonii NCTC 7869, the streptococcal species to which C. albicans bound with highest affinity, and was effective in blocking the coaggregation of C. albicans and S. gordonii cells in the fluid phase. When fixed to microtiter plate wells, the S. gordonii polysaccharide was bound by all strains of C. albicans tested. The polysaccharide contained Rha, Glc, GalNAc, GlcNAc, and Gal and was related compositionally to previously characterized cell wall polysaccharides from strains of S. oralis and S. sanguis. The adherence of yeast cells to the immobilized polysaccharide was not inhibitable by a number of saccharides. Antiserum raised to the S. gordonii NCTC 7869 polysaccharide blocked adherence of C. albicans ATCC 10261 to the polysaccharide. The results identify a complex cell wall polysaccharide of S. gordonii as the coaggregation receptor for C. albicans. Adherent interactions of yeast cells with streptococci and other bacteria may be important for colonization of both hard and soft oral surfaces by C. albicans.

Candida albicans↗
PubMed2001

Characterization of a Brucella species 25-kilobase DNA fragment deleted from Brucella abortus reveals a large gene cluster related to the synthesis of a polysaccharide.

In the present study we completed the nucleotide sequence of a Brucella melitensis 16M DNA fragment deleted from B. abortus that accounts for 25,064 bp and show that the other Brucella spp. contain the entire 25-kb DNA fragment. Two short direct repeats of four nucleotides, detected in the B. melitensis 16M DNA flanking both sides of the fragment deleted from B. abortus, might have been involved in the deletion formation by a strand slippage mechanism during replication. In addition to omp31, coding for an immunogenic protein located in the Brucella outer membrane, 22 hypothetical genes were identified. Most of the proteins that would be encoded by these genes show significant homology with proteins involved in the biosynthesis of polysaccharides from other bacteria, suggesting that they might be involved in the synthesis of a Brucella polysaccharide that would be a heteropolymer synthesized by a Wzy-dependent pathway. This polysaccharide would not be synthesized in B. abortus and would be a polysaccharide not identified until present in the genus Brucella, since all of the known polysaccharides are synthesized in all smooth Brucella species. Discovery of a novel polysaccharide not synthesized in B. abortus might be interesting for a better understanding of the pathogenicity and host preference differences observed between the Brucella species. However, the possibility that the genes detected in the DNA fragment deleted in B. abortus no longer lead to the synthesis of a polysaccharide must not be excluded. They might be a remnant of the common ancestor of the alpha-2 subdivision of the class Proteobacteria, with some of its members synthesizing extracellular polysaccharides and, as Brucella spp., living in association with eukaryotic cells.

Base Sequence↗
PubMed1981

Localization and partial characterization of soybean lectin-binding polysaccharide of Rhizobium japonicum.

Immunoelectron microscopy was combined with partial characterization of isolated exopolysaccharide to study binding of soybean lectin by Rhizobium japonicum strain USDA 138. Lectin-binding activity resided in two forms of exopolysaccharide produced during growth: an apparently very high-molecular-weight capsular form and a lower-molecular-weight diffusible form. At low-speed centrifugation, the capsular form cosedimented with cells to form a viscous, white, cell-gel complex which was not diffusible in 1% agar, and the diffusible form remained in the cell-free supernatant. Electron microscopic observation of the cell-gel complex after labeling with soybean lectin-ferritin conjugate revealed that capsular polysaccharides, frequently attached to one end of the cells, were receptors for lectin. The outer membrane of the cell bound no lectin. Various preparations of exopolysaccharide isolated from the culture supernatant were tested for lectin binding, interaction with homologous somatic antigen, and the presence of 2-keto-3-deoxyoctonate and were chromatographed in Sepharose 4B and 6B gel beds. Lectin binding was restricted to a polysaccharide component designated as lectin-binding polysaccharide. This polysaccharide, as present in the cell-free culture supernatant, was a diffusible acidic polysaccharide devoid of 2-keto-3-deoxyoctonate, with a molecular weight of 2 X 10(6) to 5 X 10(6). It was concluded that the soybean lectin-binding component of R. japonicum is an extracellular polysaccharide and not a lipopolysaccharide and that the diffusible lectin-binding polysaccharide probably differs from the very high-molecular-weight lectin-binding polysaccharide of the loose capsule (slime) only in the degree of polymerization.

Lectins↗
PubMed1993

Expression of the capsular K5 polysaccharide of Escherichia coli: biochemical and electron microscopic analyses of mutants with defects in region 1 of the K5 gene cluster.

The gene cluster of the capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three regions responsible for polymerization and surface expression have been defined (I.S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1330, 1988). Region 1 has now been sequenced, and five open reading frames (kpsEDUCS) have been defined (C. Pazzani, C. Rosenow, G. J. Boulnois, D. Bronner, K. Jann, and I. S. Roberts, J. Bacteriol. 175:5978-5983, 1993). In this study, we characterized region 1 mutants by immunoelectron microscopy, membrane-associated polymerization activity, cytoplasmic CMP-2-keto-3-deoxyoctonate (KDO) synthetase activity, and chemical analysis of their K5 polysaccharides. Certain mutations within region 1 not only effected polysaccharide transport (lack of region 1 gene products) but also impaired the polymerization capacity of the respective membranes, reflected in reduced amounts of polysaccharide but not in its chain length. KDO and phosphatidic acid (phosphatidyl-KDO) substitution was found with extracellular and periplasmic polysaccharide and not with cytoplasmic polysaccharide. This and the fact that the K5 polysaccharide is formed in a kpsU mutant (defective in capsule-specific K-CMP-KDO synthetase) showed that CMP-KDO is engaged not in initiation of polymerization but in translocation of the polysaccharide.

Antigens, Bacterial↗