Extracellular Capsular Polysaccharide Research Articles

Campylobacter jejuni and Postinfectious Autoimmune Diseases: AProof of Concept in Glycobiology.

Glycans, one of the most diverse groups of macromolecules, are ubiquitous constituents of all cells and have many critical functions, including the interaction between microbes and their hosts. One of the best model organisms to study the host-pathogen interaction, the gastrointestinal pathogen Campylobacter jejuni dedicates extensive resources to glycosylation and exhibits a diverse array of surface sugar-coated displays. The first bacterium where N-linked glycosylation was described, C. jejuni can additionally modify proteins by O-linked glycosylation, has extracellular capsular polysaccharides that are important for virulence and represent the major determinant of the Penner serotyping scheme, and has outer membrane lipooligosaccharides that participate in processes such as colonization, survival, inflammation, and immune evasion. In addition to causing gastrointestinal disease and extraintestinal infections, C. jejuni was also linked to postinfectious autoimmune neuropathies, of which Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS) are the most extensively characterized ones. These postinfectious autoimmune neuropathies occur when specific bacterial surface lipooligosaccharides mimic gangliosides in the host nervous system. C. jejuni provided the first proof of concept for the involvement of molecular mimicry in the pathogenesis of an autoimmune disease and, also, for the ability of a bacterial polymorphism to shape the clinical presentation of the postinfectious autoimmune neuropathy. The scientific journey that culminated with elucidating the mechanistic details of the C. jejuni-GBS link was the result of contributions from several fields, including microbiology, structural biology, glycobiology, genetics, and immunology and provides an inspiring and important example to interrogate other instances of molecular mimicry and their involvement in autoimmune disease.

New insight into the mechanism by which antifreeze peptides regulate the physiological function of Streptococcus thermophilus subjected to freezing stress

IntroductionAntifreeze peptides regulate the physiological functions of frozen cells and even their apoptosis; however, the mechanisms by which antifreeze peptides regulate these processes remain unclear, although the interactions between cell membranes and ice are well known to be important in this process. ObjectivesOur study aims to investigate how antifreeze peptides regulate cell physiological functions during the freezing process. MethodsWe investigated the cryoprotective effect of rsfAFP on the physiological functions of S. thermophilus under freezing stress by measuring cellular metabolism activity, intracellular enzyme activity, cell membrane characterization, and cell apoptosis. The mechanism by which rsfAFP impacts S. thermophilus physiological functions under freezing stress was investigated using multispectral techniques and cryo-TEM. ResultsWe show that a recombinant antifreeze peptide (rsfAFP) interacts with the extracellular capsular polysaccharides and peptidoglycan of Streptococcus thermophilus and ice to cover the outer layer of the membrane, forming a dense protective layer that regulates the molecular structure of extracellular ice crystals, which results in reduced extracellular membrane damage, depressed apoptosis and increased intracellular metabolic activity. This interaction mechanism was indicated by the fact that S. thermophilus better maintained its permeability barrier, membrane fluidity, membrane structural integrity, and cytoplasmic membrane potential during freezing stress with rsfAFP treatment. ConclusionThese results provide new insights into the mechanism by which rsfAFP regulates frozen cellphysiological functionsand apoptosis under freezing stress.

Synthesis of a dimer of the repeating unit of type Ia group BStreptococcusextracellular capsular polysaccharide and immunological evaluations of related protein conjugates

Type Ia group B Streptococcus (GBS) is one of the major causes of fatal infections in neonates. Its extracellular capsular polysaccharide (CPS) is a useful target for the development of anti-type Ia GBS vaccines. To explore the structure-activity relationships of type Ia GBS CPS and design more effective vaccines, a dimer of the branched pentasaccharide repeating unit of this CPS was synthesized by a highly convergent strategy highlighted by constructing the key intermediate via one-pot iterative glycosylation and imposing two side chains in one step via dual glycosylation. This represented the first total synthesis of a dimer of the repeating unit of any GBS CPS reported so far and the strategy should be applicable to higher oligomers of this repeating unit. The synthetic dimer and its monomeric analog were coupled with CRM197 carrier protein to generate conjugates that were evaluated in mice. Immunological results revealed that both carbohydrate antigens could induce robust total and IgG antibody responses and the elicited antibodies were cross-reactive with both carbohydrate antigens. It was concluded that both the monomeric and the dimeric repeating units may be employed as haptens for anti-type Ia GBS vaccine development.

Extraction and Visualization of Capsular Polysaccharide from Acinetobacter baumannii.

Methods to determine the presence of a bacterial capsule typically involve the use of stains specific for capsule, the exclusion of a dye by the capsule or by electron microscopy. However, these procedures require equipment that may not be readily available to all researchers. Here we describe a method for extraction, visualization, and quantification of extracellular capsular polysaccharide from Acinetobacter baumannii with commonly used reagents and equipment.

The capsule of Streptococcus equi ssp. zooepidemicus is a target for attenuation in vaccine development

Streptococcus equi ssp. zooepidemicus (SEZ) is an important pathogen associated with a wide range of diseases in many mammalian species. The development of novel effective vaccines would be beneficial to control SEZ infection. In the present study, the importance of the SEZ capsule was examined using a newly constructed capsule-deficient mutant ΔhasB strain. Transmission electron microscopy confirmed a decrease in the abundance of extracellular capsular polysaccharide on the mutant SEZ. Compared to the parental wild-type SEZ, the ΔhasB mutant was highly attenuated in mice and provided 100% protection against lethal challenge when administered as a live vaccine. Real-time PCR analysis showed a marked increased in the levels of IL-4 and IFN-γ mRNA in immunized mice. The role that the capsule plays in SEZ pathogenicity was also explored with respect to the mechanistic design of an attenuated vaccine target. The capsule could resist complement C3 deposition on the surface of SEZ cells and aid in preventing complement-mediated opsonization and phagocytosis by cultured macrophages. These results suggest that the capsule of SEZ plays an important role in pathogenicity and may serve as a target for attenuation in vaccine development.

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.

Cloning and mutagenesis of a serotype-specific DNA region involved in encapsulation and virulence of Actinobacillus pleuropneumoniae serotype 5a: concomitant expression of serotype 5a and 1 capsular polysaccharides in recombinant A. pleuropneumoniae serotype 1.

A DNA region involved in Actinobacillus pleuropneumoniae serotype 5 capsular polysaccharide (CP) biosynthesis was identified and characterized by using a probe specific for the cpxD gene involved in CP export. The adjacent serotype 5-specific CP biosynthesis region was cloned from a 5.8-kb BamHI fragment and an 8.0-kb EcoRI fragment of strain J45 genomic DNA. DNA sequence analysis demonstrated that this region contained four complete open reading frames, cps5A, cps5B, cps5C, and cps5D. Cps5A, Cps5B, and Cps5C showed low homology with several bacterial glycosyltransferases involved in the biosynthesis of lipopolysaccharide or CP. However, Cps5D had high homology with KdsA proteins (3-deoxy-D-manno-2-octulosonic acid 8-phosphate synthetase) from other gram-negative bacteria. The G+C content of cps5ABC was substantially lower (28%) than that of cps5D and the rest of the A. pleuropneumoniae chromosome (42%). A 2.1-kb deletion spanning the cloned cps5ABC open reading frames was constructed and transferred into the J45 chromosome by homologous recombination with a kanamycin resistance cassette to produce mutant J45-100. Multiplex PCR confirmed the deletion in this region of J45-100 DNA. J45-100 did not produce intracellular or extracellular CP, indicating that cps5A, cps5B, and/or cps5C were involved in CP biosynthesis. However, biosynthesis of the Apx toxins, lipopolysaccharide, and membrane proteins was unaffected by the mutation. Besides lack of CP biosynthesis, and in contrast to J45, J45-100 grew faster, was sensitive to killing in precolostral calf serum, and was avirulent in pigs at an intratracheal challenge dose three times the 50% lethal dose (LD50) of strain J45. At six times the J45 LD50, J45-100 caused mild to moderate lung lesions but not death. Electroporation of cps5ABC into A. pleuropneumoniae serotype 1 strain 4074 generated strain 4074(pJMLCPS5), which expressed both serotype 1 and serotype 5 CP. However, serotype 1 capsule expression was diminished in 4074(pJMLCPS5) in comparison to 4074. The recombinant strain produced significantly less total CP (serotypes 1 and 5 CP combined) in log phase (P = 0.0012) but significantly more total CP in late stationary phase than 4074 (P < 0.0001). In addition, strain 4074(pJMLCPS5) caused less mortality and bacteremia in pigs and mice following respiratory challenge than strain 4074, indicating that virulence was affected by diminished capsule production. These results emphasize the importance of CP in the serum resistance and virulence of A. pleuropneumoniae.

An enzyme-linked immunosorbent assay for the detection of soluble Klebsiella pneumoniae capsular polysaccharide

Extracellular capsular polysaccharide (CPS) produced by Klebsiella pneumoniae capsular type 2 was quantitated by means of an enzyme-linked immunosorbent assay. Microtiter plates were coated with a CPS-specific mouse monoclonal antibody which served to capture CPS present in culture supernatants and biological fluids. A polyclonal rabbit IgG preparation with high antibody content against CPS type 2 was added as a secondary antibody, and bound rabbit IgG was visualized by the addition of alkaline-phosphatase-conjugated anti-rabbit-IgG. Simultaneous testing of standards of purified CPS type 2 permitted the quantitation of CPS in samples of culture fluid, mouse and human plasma, and human urine. Due to its high sensitivity with a lower limit of detectability of 1 ng·ml −1 of CPS, the test system may prove superior to standard chemical methods of CPS quantification.

Visualization of capsular polysaccharide of coagulase negative staphylococci (CNS) associated with vascular prosthetic graft material

The coagulase negative staphylococci (CNS) are a ubiquitous group of microorganisms which comprise a major component of the microbial flora of the skin and adjacent glands. Historically, the CNS were viewed as insignificant contaminants with little clinical importance. However, over the last decade these organisms have emerged as important etiologic agents of biomaterial associated infections. Furthermore, many of the Staphylococcus epidermidis strains recovered from catheter or prosthetic infections exhibit the capacity to produced an extracellular capsular polysaccharide (slime) which is viewed as an important factor in biomaterial adherence and persistence. Bacterial capsular material is composed of hydrated polysaccharides and has been visualized by transmission electron microscopy when lysine is added to the fixation protocol. This study reports on the use of a lysine-ruthenium redglutaraldehyde fixation technique for visualizing the polysaccharide capsule on two coagulase negative staphylococcal reference strains, S. epidermidis RP62A (ATCC 35984) and S. hominis SP2 (ATCC 35982).