Streptococcus iniae capsule impairs phagocytic clearance and contributes to virulence in fish

Surface capsular polysaccharides play a critical role in protecting several pathogenic microbes against innate host defenses during infection. Little is known about virulence mechanisms of the fish pathogen Streptococcus iniae, though indirect evidence suggests that capsule could represent an important factor. The putative S. iniae capsule operon contains a homologue of the cpsD gene, which is required for capsule polymerization and export in group B Streptococcus and Streptococcus pneumoniae. To elucidate the role of capsule in the S. iniae infectious process, we deleted cpsD from the genomes of two virulent S. iniae strains by allelic exchange mutagenesis to generate the isogenic capsule-deficient DeltacpsD strains. Compared to wild-type S. iniae, the DeltacpsD mutants had a predicted reduction in buoyancy and cell surface negative charge. Transmission electron microscopy confirmed a decrease in the abundance of extracellular capsular polysaccharide. Gas-liquid chromatography-mass spectrometry analysis of the S. iniae extracellular polysaccharides showed the presence of l-fucose, d-mannose, d-galactose, d-glucose, d-glucuronic acid, N-acetyl-d-galactosamine, and N-acetyl-d-glucosamine, and all except mannose were reduced in concentration in the isogenic mutant. The DeltacpsD mutants were highly attenuated in vivo in a hybrid striped bass infection challenge despite being more adherent and invasive to fish epithelial cells and more resistant to cationic antimicrobial peptides than wild-type S. iniae. Increased susceptibility of the S. iniae DeltacpsD mutants to phagocytic killing in whole fish blood and by a fish macrophage cell line confirmed the role of capsule in virulence and highlighted its antiphagocytic function. In summary, we report a genetically defined study on the role of capsule in S. iniae virulence and provide preliminary analysis of S. iniae capsular polysaccharide sugar components.     

Conjugation of capsular polysaccharide to alpha-haemolysin from Staphylococcus aureus as a glycoprotein antigen

The capsular polysaccharides of two pathogenic strains of Staphylococcus aureus 8914 and 31 were isolated and purified. These polysaccharides were conjugated to alpha-haemolysin prepared from the same strains. Amongst the various coupling procedures which were tested the best results were obtained with sodium cyanoborohydride and glutaraldehyde. The conjugates were purified and their immunologic properties were tested. They gave a positive response against antisera from whole bacterial cells.     

Knockout mutagenesis of the kpsE gene of Campylobacter jejuni 81116 and its involvement in bacterium-host interactions

Campylobacter jejuni is a common cause of bacterial enteritis. The surface capsular polysaccharides are important for this bacterium to survive in the environment, but little is known about their involvement in bacterium-host interactions. This study showed that the C. jejuni capsular polysaccharides play an important role in adherence to and invasion of human embryonic epithelial cells. However, no significant role of capsular polysaccharides was shown in colonization of the chicken gut.     

革兰氏阳性菌荚膜多糖研究进展*

细菌的荚膜多糖是生物膜的重要组成部分,在细菌的生长分裂、维持细胞壁形态、抵御外界环境以及免疫反应等方面都起到重要作用。在致病菌中,荚膜多糖常作为一种毒力因子发挥作用。在革兰氏阳性菌中,荚膜多糖的化学结构、生物合成过程及功能应用越来越受到关注。讨论了革兰氏阳性菌中部分致病菌的荚膜多糖与非致病菌表面多糖的分布位置、化学组成及其结构特异性。重点讨论三种具有代表性的革兰氏阳性致病菌及非致病菌株:肺炎链球菌(Streptococcus pneumonia)、金黄色葡萄球菌(Staphylococcus aureus)及乳酸乳球菌(Lactococcus lactis)。综述革兰氏阳性菌中荚膜多糖生物合成的三种方式:Wzx/Wzy-依赖通路、ABC转运蛋白(ABC transporter)途径及合酶依赖途径,并举例解释了相应多糖的合成过程及相关基因。介绍了革兰氏阳性菌荚膜多糖及表面多糖的生理功能,如屏障保护功能、胞间黏附功能以及参与宿主细胞的免疫反应等。结合荚膜多糖的生物学功能,概述其当前主要研究进展,如构建高耐受工程菌疫苗研制等。结合细菌荚膜多糖的特征差异,对其在医药与工业生产领域的广阔前景提出展望和建议。     

Root Hair Deformations Associated with Fractionated Extracts from Rhizobium trifolii

Components from culture fluid and whole cells of Rhizobium trifolii were examined for effects on root hair morphology of white clover seedlings (Trifolium repens var. Ladino). Cell-free culture fluid, exopolysaccharides, supernatant fluid from the precipitation of the exopolysaccharides, capsular polysaccharides, lipopolysaccharides, and a protein fraction from culture fluids were assayed for morphogenetic effects on the root hairs of axenically grown clover seedlings. Crude fractions were chromatographed on Bio Gel A-5m (Bio-Rad Laboratories), and fractions collected were similarly assayed. Hexose, uronic acid, and protein concentrations were determined for all fractions assayed. Gel chromatography indicated the materials with deforming ability to be of high molecular weight (>10,000). For all fractions except exopolysaccharide, deforming ability was associated with a protein component. This suggested that two components were associated with deformation; both contained polysaccharides and one contained protein. Crude fractions differed in their ability to cause deformations and indicated the following relative ability (in decreasing order) to deform root hairs: cell-free culture fluid, capsular polysaccharides, protein from culture fluids, exopolysaccharide, and cell envelope. Lipopolysaccharides had no effect.     

Elaboration of both the group W135 and group Y capsular polysccharides by a single strain of Neisseria meningitidis

A single strain (8021) of Neisseria meningitidis, isolated from a child with disseminated meningococcal disease, was found to elaborate two serogroup-specific capsular polysaccharides-Y and W135. The original isolate as well as the progeny of ten single colony sub-isolates each agglutinated with both group Y and group W135 serogrouping antisera. The capsular polysccharide of strain 8021 contained the chemical constituents of both the W135 and Y capsular polysaccharides in a ratio of about 2.5:1. The patient responded immunologically to both capsular polysaccharides with haemagglutinating antibodies. Analysis by double diffusion in agar revealed that the capsular polysaccharide of strain 8021 contained individual molecules of group W135 and group Y capsular polysaccharides as well as a mosaic molecule containing both antigenic determinants.     

Sedimentation and viscosity studies on the capsular and somatic polysaccharides of pneumococcus Type III

Sedimentation constants at infinite dilution have been found to be 1.89 and 4.06 for the somatic and capsular polysaccharides, respectively, from pneumococcus Type III. Intrinsic viscosities have been determined for the somatic and capsular polysaccharides of pneumococcus Type III using the Ostwald viscometer. Molecular weights and dimensions have been calculated for the somatic and capsular polysaccharides of pneumococcus Type III assuming the molecules to be prolate ellipsoids of revolution. Values for the somatic polysaccharide are: molecular weight, 26,400; diameter, 0.97 mmicro; and length, 36.18 mmicro. Values for the capsular polysaccharide are: molecular weight, 171,800; diameter, 1.04 mmicro; and length, 177.87 mmicro. The molecular weights were calculated for the somatic and capsular polysaccharides of pneumococcus Type III assuming the molecules to be flexible chains. The value of the molecular weight of the somatic polysaccharide is 31,500 and the value for the molecular weight of the capsular polysaccharide is 267,500. The molecules of both the somatic and capsular polysaccharides exhibit high degrees of asymmetry.     

Fractionation of the beta-Linked Glucans of Bradyrhizobium japonicum and Their Response to Osmotic Potential

Bradyrhizobium japonicum USDA 110 synthesized both extracellular and periplasmic polysaccharides when grown on mannitol minimal medium. The extracellular polysaccharides were separated into a high-molecular-weight acidic capsular extracellular polysaccharide fraction (90% of total hexose) and three lower-molecular-weight glucan fractions by liquid chromatography. Periplasmic glucans, extracted from washed cells with 1% trichloroacetic acid, gave a similar pattern on liquid chromatography. Linkage analysis of the major periplasmic glucan fractions demonstrated mainly 6-linked glucose (63 to 68%), along with some 3,6- (8 to 18%), 3- (9 to 11%), and terminal (7 to 8%) linkages. The glucose residues were beta-linked as shown by H-nuclear magnetic resonance analysis. Glucan synthesis by B. japonicum cells grown on mannitol medium with 0 to 350 mM fructose as osmolyte was measured. Fructose at 150 mM or higher inhibited synthesis of periplasmic and extracellular 3- and 6-linked glucans but had no effect on the synthesis of capsular acidic extracellular polysaccharides.     

细菌荚膜多糖的化学结构

荚膜是一些细菌所具有的表层结构,与多种疾病有着密切联系。细菌荚膜多糖不仅结构复杂,而且在免疫活性方面发挥着重要的作用。同一种细菌根据其荚膜多糖的抗原性不同可分为不同的血清型,不同血清型细菌荚膜多糖的化学结构也存在差异。以荚膜多糖为基础的疫苗正在积极研究开发当中,对不同致病细菌荚膜多糖具体化学结构的掌握是疫苗得到许可的必备条件之一。本文对致病细菌荚膜多糖的化学结构进行了归纳和总结,以期为荚膜多糖的化学结构研究和疫苗开发提供参考。     

SEDIMENTATION AND VISCOSITY STUDIES ON THE CAPSULAR AND SOMATIC POLYSACCHARIDES OF PNEUMOCOCCUS TYPE III

Sedimentation constants at infinite dilution have been found to be 1.89 and 4.06 for the somatic and capsular polysaccharides, respectively, from pneumococcus Type III. Intrinsic viscosities have been determined for the somatic and capsular polysaccharides of pneumococcus Type III using the Ostwald viscometer. Molecular weights and dimensions have been calculated for the somatic and capsular polysaccharides of pneumococcus Type III assuming the molecules to be prolate ellipsoids of revolution. Values for the somatic polysaccharide are: molecular weight, 26,400; diameter, 0.97 mµ; and length, 36.18 mµ. Values for the capsular polysaccharide are: molecular weight, 171,800; diameter, 1.04 mµ; and length, 177.87 mµ. The molecular weights were calculated for the somatic and capsular polysaccharides of pneumococcus Type III assuming the molecules to be flexible chains. The value of the molecular weight of the somatic polysaccharide is 31,500 and the value for the molecular weight of the capsular polysaccharide is 267,500. The molecules of both the somatic and capsular polysaccharides exhibit high degrees of asymmetry.     

Human memory B lymphocyte subsets fulfill distinct roles in the anti-polysaccharide and anti-protein immune response

There is controversy on the role of IgM memory and switched memory B lymphocytes in the Ab response to T cell-independent and T cell-dependent Ags. We transplanted SCID/SCID mice with human B lymphocyte subsets and immunized them with heat-inactivated Streptococcus pneumoniae or with a pneumococcal vaccine. Inactivated S. pneumoniae and soluble pneumococcal capsular polysaccharides elicited an IgM anti-polysaccharide and anti-protein Ab response from IgM memory B lymphocytes and an IgG anti-polysaccharide and anti-protein response from switched memory B lymphocytes. In addition to the IgM Ab response, IgM memory B cells elicited an IgG anti-polysaccharide and anti-protein Ab response after immunization with inactivated S. pneumoniae or soluble pneumococcal capsular polysaccharides. In conclusion, our findings provide evidence for a versatile role of IgM memory B cells in T-independent and T-dependent immune responses.     

Capsular antigens of bacteria. Capsular antigens as the basis of vaccines against pathogenic bacteria

The role of bacterial capsular antigens represented in capsular polysaccharides and exoglycans in pathogenicity and virulence of bacteria is discussed in this review. Using capsular antigens for vaccines against severe diseases caused by capsular microorganisms is considered in detail. The use of conjugates of capsular polysaccharides and their fragments with proteins and peptides for vaccine as well as using liposomes as adjuvants for the capsular antigens are described. Data concerning structural elucidation of bacterial capsular antigens are given in the first part of this review. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 9, pp. 1175–1182.     

The capsular polysaccharide of Bacteroides fragilis comprises two ionically linked polysaccharides.

Recently, we have shown that the capsular polysaccharide of Bacteroides fragilis NCTC 9343 is composed of an aggregate of two discrete large molecular weight polysaccharides (designated polysaccharides A and B). Following disaggregation of this capsular complex by very mild acid treatment, high resolution NMR spectroscopy demonstrated that polysaccharides A and B consist of highly charged repeating unit structures with unusual substituent groups (Baumann, H., Tzianabos, A. O., Brisson, J.-R., Kasper, D.L., and Jennings, H.J. (1992) Biochemistry 31, 4081-4089). Presently, we report that the capsular polysaccharide of B. fragilis represents a complex structure that is formed as a result of ionic interactions between polysaccharides A and B. Electron microscopy of immunogold-labeled organisms (with monoclonal antibodies specific for polysaccharides A and B) demonstrated that the two polysaccharides are co-expressed on the cell surface of B. fragilis. We have shown that the purified capsule complex is made up exclusively of polysaccharide A and polysaccharide B (no other macromolecular structure was detected) in a 1:3.3 ratio and that disaggregation of this complex into the native forms of the constituent polysaccharides could be accomplished by preparative isoelectric focusing. Structural analyses of the native polysaccharides A and B showed that they possessed the same repeating unit structures as the respective acid-derived polysaccharides. The ionic nature of the linkage between polysaccharides A and B was demonstrated by reassociation of the native polysaccharides to form an aggregated polymer comparable to the original complex. The distinctive composition of this macromolecule may provide a rationale for the unusual biologic properties associated with the B. fragilis capsular polysaccharide.     

Extracellular polysaccharide production by Klebsiella pneumoniae and its relationship to virulence

Klebsiella pneumoniae serotype 1 and serotype 2 and their capsular variants were examined for production of cell-associated capsular polysaccharides and extracellular capsular polysaccharides. The virulence of these organisms in experimental animals was examined via intraperitoneal injection in mice and transtracheal inoculation into the lungs of rats. It was found that the production of either polysaccharide component correlated with the observed virulence. The extracellular polysaccharides were purified by ethanol precipitation, electrodialysis, extraction with quaternary ammonium salts, and gel filtration. These purification steps allowed for the separation and purification of both the extracellular lipopolysaccharide and the extracellular capsular polysaccharide. Purified extracellular capsular polysaccharide and extracellular lipopolysaccharide were co-injected with K. pneumoniae intraperitoneally into mice to determine if either of these substances would produce an effect on the natural course of infection in these animals. These studies showed that only purified extracellular lipopolysaccharide enhanced the virulence of K. pneumoniae when co-injected into mice, and this virulence enhancement correlated with the content of extracellular lipopolysaccharide, but not extracellular capsular polysaccharide in mixtures of these polysaccharides. Saponification of K. pneumoniae serotype 1 extracellular polysaccharides significantly decreased their virulence-enhancing capabilities in mice, further suggesting that extracellular lipopolysaccharide may play a role in these infections.     

Classification ofVibrio vulnificusStrains by the Carbohydrate Composition of Their Capsular Polysaccharides

Pathogenic bacteria are often classified on the basis of the complex polysaccharides found on the surface, usually capsular polysaccharides or lipopolysaccharides. It is common in clinical practice to use reactivity with antisera specific to the various cell surface carbohydrates for this purpose. In this work, we describe a chemotyping method for bacterial capsular polysaccharides which is based on a carbohydrate analysis of an acid hydrolysate of the capsule. High-performance anion-exchange chromatography at high pH (HPAE) with electrochemical detection, which is used for analysis of the hydrolysate, shows preferential sensitivity for sugars. A single acid hydrolysis condition is chosen for screening a large collection of bacterial isolates and a computerized autosampler is used to make possible a large number of rapid analyses. This procedure does not yield a quantitative carbohydrate analysis for the sample but produces a fingerprint which can be used to discriminate among isolates which have different capsular polysaccharide structures. The procedure has been applied to a collection of 120 isolates ofVibrio vulnificus,a water-born species common in shellfish which causes septicemia in immunocompromised individuals, most often from eating of raw oysters. The collection of bacterial isolates includes strains from both clinical cases of septicemia and from such environmental sources such as sea water, sediments, and shellfish. Our results show that a number of unusual sugars including many amino sugars are found in these polysaccharides and that a wide variety of capsular carbotypes inV. vulnificusmay be readily distinguished by the HPAE fingerprint.     

Structure analysis of the Staphylococcus aureus UDP-N-acetyl-mannosamine dehydrogenase Cap5O involved in capsular polysaccharide biosynthesis

Bacterial UDP-sugar dehydrogenases are part of the biosynthesis pathway of extracellular polysaccharides. These compounds act as important virulence factors by protecting the cell from opsonophagocytosis and complement-mediated killing. In Staphylococcus aureus, the protein Cap5O catalyzes the oxidation of UDP-N-acetyl-mannosamine to UDP-N-acetyl-mannosaminuronic acid. Cap5O is crucial for the production of serotype 5 capsular polysaccharide that prevents the interaction of bacteria with both phagocytic and nonphagocytic eukaryotic cells. However, details of its catalytic mechanism remain unknown. We thus crystallized Cap5O and solved the first structure of an UDP-N-acetyl-mannosamine dehydrogenase. This study revealed that the catalytic cysteine makes a disulfide bond that has never been observed in other structurally characterized members of the NDP-sugar dehydrogenase family. Biochemical and mutagenesis experiments demonstrated that the formation of this disulfide bridge regulates the activity of Cap5O. We also identified two arginine residues essential for Cap5O activity. Previous data suggested that Cap5O is activated by tyrosine phosphorylation, so we characterized the phosphorylation site and examined the underlying regulatory mechanism.     

The Fate of Sulphate within the Capsular Polysaccharide of the Unicellular Red Alga Rhodella maculata

³⁵SO^2–₄ was used in pulse-chase experiments to study themetabolism of sulphate in the capsular polysaccharides of R.maculata. Radioactivity reached an equilibrium between the chiefintracellular pools during an 8 h pulse and there was no lossof radioactivity from the cells during an extended chase ofup to 14 d, until cultures reached stationary growth phase.Any loss then found was due to dissociation of sulphated capsularmaterial from the cells. The failure of biochemical assays todetect extracellular sulphatase or sulphotransferase activitiessupports the conclusion from pulse-chase experiments that thereis no turnover of sulphate in the capsular polysaccharide ofR. maculata. Key words: sulphated polysaccharides, sulphate turnover, Rhodella     

Occurrence of a capsule in Aeromonas salmonicida

Aeromonas salmonicida grown in a medium with excess glucose as carbon source produces both capsular and exocellular polysaccharides. The capsular polysaccharide is composed of glucose, mannose, rhamnose, N-acetylmannosamine and mannuronic acid in the molar ratios of approximately 5:3:0.75:2:1. The extracellular polysaccharide is similarly constituted, but in the molar ratios of approximately 4.75:10.5:1.5:2:1. The capsular and exocellular polysaccharides did not cross-react with monoclonal antibodies against the A-layer or the O-antigen lipopolysaccharide.     

Antigenic identity of the capsule lipopolysaccharides,exopolysaccharides, and O-specific polysaccharides in Azospirillum brasilense

The antigenic identity (and close values of electrophoretic mobility) of capsular polysaccharides, exopolysaccharides, and O-specific polysaccharides was revealed in the Azospirillum brasilense strains Sp7 and Sp245 by the immunodiffusion and immunoelectrophoretic methods. Together with the literature data on the identity of the monosaccharides composition of these polymers, this gives evidence of the absence of a specific capsular antigen in the bacteria studied. Thus, extracellular Azospirillum brasilense polysaccharides are likely to represent O-antigenic lipopolysaccharide fragments excreted by the bacteria into the culture medium, and their identification as a capsule or as an exopolysaccharide depends on the strength of the attachment of these polysaccharides to the cell surface.     

Structural investigation of the capsular polysaccharide from Escherichia coli O9:K37 (A 84a)

The structure of the capsular polysaccharide from E. coli O9:K37 (A 84a) has been studied, using methylation analysis, Smith degradation, and graded acid hydrolysis. The configurations at the anomeric centres were assigned by 1H-n.m.r. spectroscopy of the polysaccharide and its derivatives and oligosaccharide fragments. The polysaccharide has the following trisaccharide repeating-unit which is unique in the E. coli series of capsular polysaccharides in possessing a 1-carboxyethylidene group as the sole acidic function. (Formula: see text) E. coli capsular polysaccharides have been classified into seventy-four serotypes. The structures of about twenty of these polysaccharides have been elucidated, one of which, K29, has been reported to contain a 1-carboxyethylidene group. In continuation of a programme aimed at establishing the structural basis for the serology and immunochemistry of the E. coli capsular antigens, we now report on the structure of the capsular polysaccharide from E. coli O9:K37.