Molecular characterization of the complete 23F capsular polysaccharide locus of Streptococcus pneumoniae

The complete DNA sequence of the capsular locus 23F of Streptococcus pneumoniae is presented. The 18.6-kb cps23f locus is composed of 18 open reading frames flanked at the 5' and 3' ends by the genes dexB and aliA, an arrangement similar to those of some of the other identified cps loci.     

Functional analysis of glycosyltransferases encoded by the capsular polysaccharide biosynthesis locus of Streptococcus pneumoniae serotype 14

Bacteria belonging to the species Streptococcus pneumoniae vary in their capsule. Presently, 90 capsular serotypes are known, all possessing their own specific polysaccharide structure. Little is known about the biosynthesis of these capsular polysaccharides. The cps locus of S. pneumoniae serotype 14 was cloned. So far, 7 open reading frames have been sequenced, cps14B to cps14H. The gene products are similar to proteins involved in bacterial polysaccharide biosynthesis, both of Gram-negative and -positive micro-organisms. Gene-specific mutants were created for cps14D to cps14H by insertional mutagenesis. All mutants no longer agglutinated with a monoclonal antibody against type 14 capsule polysaccharides. The biosynthetic function of cps14E and cps14G was determined by analysis of the intermediates in the synthesis of the oligosaccharide subunit, formed in membrane preparations of the wild-type and mutant strains and in membrane preparations of Escherichia coli expressing the pneumococcal glycosyltransferases. The enzyme encoded by cps14E is a glucosyl-1-phosphate transferase that links glucose to a lipid carrier, the first step in the biosynthesis of the type 14 repeating unit. The gene product of cps14G encodes a beta-1,4-galactosyltransferase, the enzyme responsible for the second step in the subunit synthesis, the transfer of galactose to lipid-linked glucose.     

Synthesis of four spacer-containing 'tetrasaccharides' that represent four possible repeating units of the capsular polysaccharide of Streptococcus pneumoniae type 6B

In the framework of studies towards oligosaccharide-conjugate based vaccines against Streptococcus pneumoniae, the synthesis is reported of four spacer-containing tetrasaccharides that each can be conceived as representing a repeating unit of the capsular polysaccharide of S. pneumoniae serotype 6B, namely, 3-aminopropyl D-ribityl-(5-->hydrogen phosphate-->2)-alpha-D-galactopyranosyl-(1-->3) -alpha-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranoside, 3-aminopropyl alpha-L-rhamnopyranosyl-(1-->4)-D-ribityl-5(-->hydrogen phosphate-->2)-alpha-D-galactopyranosyl-(1-->3)-alpha-D-glucopyranoside, 3-aminopropyl alpha-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl-(1-->4) -D-ribityl-(5-->hydrogen phosphate-->2) -alpha-D-galactopyranoside, and alpha-D-galactopyranosyl-(1-->3)-alpha-D-glucopyranosyl-(1-->3)-alpha-L -rhamnopyranosyl-(1-->4)-5-O-(3-aminopropyl hydrogen phosphate)-D-ribitol. Phosphorylations were carried out using the H-phosphonate method.     

Antibody responses to capsular polysaccharide backbone and O-acetate side groups of Streptococcus pneumoniae type 9V in humans and rhesus macaques

Streptococcus pneumoniae is responsible for high rates of pneumococcal bacteremia, meningitis, pneumonia, and acute otitis media worldwide. Protection from disease is conferred by antibodies specific for the polysaccharide (Ps) capsule of the bacteria. Of the four types of group 9 pneumococci, types 9N and 9V cause the most disease, and both types are included in the polyvalent pneumococcal vaccine. The type 9V capsule consists of repeating pentasaccharide units linearly arranged, with an average of 1 to 2 mol of O-acetate side chains per mol of repeat units, added in a complex pattern in which not all repeat units are alike. alpha-GlcA residues may be O-acetylated in the 2 (17%) or 3 (25%) position and beta-ManNAc residues may be O-acetylated in the 4 (6%) or 6 (55%) position. Under certain conditions, the O-acetate side chains are subject to oxidation, which results in subsequent de-O-acetylation of a significant number of the repeat units. This de-O-acetylation could adversely affect the efficacy of a vaccine containing the 9V Ps. A study was undertaken to compare the relative contributions of O-acetate and Ps backbone epitopes in the immune response to S. pneumoniae 9V type-specific Ps. In both an infant rhesus monkey model and humans, antibodies against the non-O-acetylated 9V backbone as well as against O-acetylated 9V Ps were detected. Functional (opsonophagocytic) activity was observed in antisera in which the predominant species of antibody recognized de-O-acetylated 9V Ps. We concluded that the O-acetate side groups, while recognized, are not essential to the ability of the 9V Ps to induce functional antibody responses.     

Spread of the serotype 23F multidrug-resistant Streptococcus pneumoniae clone to South Korea

We report the case of 61-year-old woman with cryptogenic liver abscesses who had been profoundly ill with severe upper abdominal pain, impaired consciousness, prostration, continuous high fever secondary to sepsis, and thrombocytopenia (platelets, 1-5 x 10(4)/mm3) since admission. Ultrasonograms and computed tomograms revealed two separate multiloculated lesions in the right lobe of the liver, consistent with the liver abscesses. Immediately after diagnosis, percutaneous abscess drainage was performed under ultrasonographic guidance; however, only a small amount of pus was drained, prompting continuous irrigation of the abscess cavity. Four days later, transcatheter hepatic arterial infusion of antibiotics was attempted. However, the abscesses had enlarged and her general condition had worsened. On hospital day 8, she underwent right hepatectomy because the multiloculated lesions were refractory to drainage. The operation was successful in terms of hepatectomy, although she continued to suffer from sepsis, secondary right subphrenic abscess formation, and prolonged thrombocytopenia with associated coagulation disorders for two months. Examination of multiple cross sections of the resected specimen disclosed that the lesions consisted of aggregations of multiple small locules. There was no communication between the locules and there were true septations, rather than multiloculated lesions with pseudoseptations. The patient has been well for 2 years without recurrent abscess of the liver or any infectious disease.     

The chemical structures of the capsular polysaccharides from Streptococcus pneumoniae types 32F and 32A

The structures of the capsular polysaccharides from Streptococcus pneumoniae types 32F and 32A have been determined by means of NMR spectroscopy as the principal method. It is concluded that both polysaccharides are composed of tetrasaccharide repeating units with a phosphorylcholine (PCho) group linked to the 3-position of the 4-substituted beta-L-rhamnose (Rha) residue. Both polysaccharides are substituted with one O-acetyl group at the 2-position of the same beta-L-rhamnose residue. In addition, the type-32A polysaccharide is substituted with another O-acetyl group at the 4-position of the 2,3-disubstituted alpha-D-glucose residue, i.e. the branch-point residue. An unusual detail in the structure is that the side chain is composed of a rhamnosyl phosphate. [chemical structure: see text] In the type-32F polysaccharide R=H, and in the type-32A polysaccharide R=Ac. The structure of C-polysaccharide found in our preparations of type-32F and type-32A capsular polysaccharides is in agreement with that published previously for the pneumococcal common antigen C-polysaccharide [Fischer, W., Behr, T., Hartmann, R., Peter-Katalinic, J. & Egge, H. (1993) Eur. J. Biochem. 215, 851-857; Kulakowska, M., Brisson, J.-R., Griffith, D. W., Young, N. M. & Jennings, H. J. (1993) Can. J. Chem. 71, 644-648].     

Detection of Streptococcus pneumoniae in clinical specimens, its antimicrobial susceptibility and serotypes

The purpose of our investigation was the detection of S. pneumoniae and the isolation frequency of PRSP/PISP from the patients of the area. 457 strains of S. pneumoniae were isolated from the clinical specimens which had been requested for cultivation from the individual general practitioners in the city. Screening of the PRSP/PISP in 457 strains was carried out with the oxacillin (MPIPC) disk method. The MIC (benezylpenicillin: PCG) was measured at the same time in 73 strains, and compared with the disk method. The results were as follows: 1) Regarding the detection rate of S. pneumoniae, nose discharge was the highest (11.4%) in all the specimens. 2) There was a greater number of isolation of S. pneumoniae in one year from November to March. Then it was scarce in August and September. This change in seasons was noted. 3) There were many 9-year olds and over-60 year olds in the age group of the patients in whom the S. pneumoniae was isolated. 4) All 30 strains where a 6 mm diameter was found when a MIC was compared with the MPIPC disk method were resistant (PRSP/PISP). All 20 strains with a > or = 20 mm diameter were sensitive (PSSP). But in the 23 strains which had a 7 mm -19 mm, 7 strains (30%) were PSSP, and 16 strains (70%) in the intermediate (PISP). 5) A diameter of 6 mm was found in 194 strains (42.5%) with the MPIPC disk method, among the 457 strains isolated from the clinical specimens. 141 strains (30.9%) were found with a diameter of 7 mm -19 mm and 122 strains (26.7%) with a diameter of > or = 20 mm. 6) More than 70% in PRSP/PISP were multiple antimicrobiotics resistant strains to PCG, EM and MINO. 7) The distribution of the serotype in PRSP/PISP was in the order of 19 type (41.3%), 23 type (21.7%), 6 type (13.0%). Moreover, it was in the order of 3 type (18.5%), 6 type (11.1%), 19 type (11.1%), and there were few 23 type (3.7%) for PSSP. The distribution of the serotype was different for PRSP/PISP and PSSP.     

Association of intrastrain phase variation in quantity of capsular polysaccharide and teichoic acid with the virulence of Streptococcus pneumoniae

The pneumococcus undergoes spontaneous phase variation between an opaque and a transparent colony form. In an animal model of systemic infection following intraperitoneal inoculation of mice, the opaque phenotype was significantly more virulent than the transparent for each of 3 strains examined. The opaque phenotype was associated with 1.2- to 5.6-fold greater amounts of capsular polysaccharide compared with the transparent using a sandwich ELISA. A similar technique comparing the amount of total teichoic acid showed that the transparent phenotype had 2.1- to 3.8-fold more immunodetectable teichoic acid. This difference was confirmed by comparing the incorporation of [3H]choline into teichoic acid. Cell fractionation revealed that variation in quantity of incorporated choline was due to differences in cell wall-associated teichoic acid. Results suggest that the pneumococcus phase varies between a virulent form with more capsular polysaccharide and less teichoic acid and an avirulent form with less capsular polysaccharide and more teichoic acid.     

NMR assignment and conformational analysis of the antigenic capsular polysaccharide from Streptococcus pneumoniae type 9N in aqueous solution

Complete 1H and 13C NMR assignments, determined by one- and two-dimensional homo- and hetero-nuclear experiments, are reported for the antigenic capsular polysaccharide (CPS) from Streptococcus pneumoniae serotype 9N (S9 in American nomenclature). Distance constraints derived from 1D NOE difference experiments were combined with energy minimisation (simulated annealing) and molecular dynamics (MD) calculations to determine the most favoured conformation of S9 in aqueous solution at 70 degrees C. NOE values simulated for several static conformational models using the NOEMOL program did not correlate well with experimental values, whereas time averaged interproton distances calculated from 500 ps of restrained MD (using the Tropp formalism to account for rapid internal mobility) were in close agreement with experimentally derived distance estimates.     

Evidence for serum immunoglobulin G (IgG) antibody responses in Macaca fascicularis identified by monoclonal antibodies to human IgG subclasses

This investigation determined the capacity of murine monoclonal antibodies directed to human immunoglobulin G (IgG) subclasses to identify molecules with conserved epitopes in the serum of the nonhuman primate, Macaca fascicularis. We subsequently utilized this cross-reactivity to document the characteristics of IgG subclass antibody responses in M. fascicularis to parenteral immunization with intact oral microorganisms, antigens from oral microorganisms, and finally a defined protein toxin, tetanus toxoid. The IgG response in nonhuman primates immunized with tetanus toxoid showed a 40-fold and 110-fold increase after primary and secondary immunizations, respectively. The major IgG subclass responses were IgG1 and IgG3, with little, though significant, responses in the IgG4 and IgG2 subclasses. Seventy-five to 94% of the natural IgG antibody in nonhuman primate sera to Porphyromonas gingivalis, Prevotella intermedia and Campylobacter rectus was IgG1. IgG2 and IgG3 predominated to Bacteroides fragilis, IgG4 to Actinomyces viscosus and an equal distribution among the subclasses was noted in response to Fusobacterium nucleatum. Parenteral immunization of nonhuman primates with intact P. gingivalis elicited primarily IgG3 and IgG4, while the post-immunization IgG response to P. intermedia was largely IgG1. Nonhuman primates were also parenterally immunized with cell envelope antigens of P. gingivalis, P. intermedia, or a combination of cell envelope antigen from C. rectus and F. nucleatum and cell wall antigens of A. viscosus. The greatest IgG antibody response seen post-immunization was reactive with anti-human IgG1 for all of these antigens except to C. rectus which bound nonhuman primate antibody reactive with anti-human IgG2. It appears that the bacteria and their products exhibit unique differences in their induction of serum IgG subclass antibody responses. The characteristics of their immunogenicity as detected by the nonhuman primate may contribute to the ability of the immune responses to effectively interact with these pathogens.     

Differences in virulence for mice among Streptococcus pneumoniae strains of capsular types 2, 3, 4, 5, and 6 are not attributable to differences in pneumolysin production

We observed that differences in the in vivo growth kinetics of pneumococcal strains of capsular types 3, 4, 5, and 6 were reminiscent of differences that we had previously reported for type 2 strain D39 and its pneumolysin-deficient mutant, PLN. Capsular type 2 Streptococcus pneumoniae D39 exhibits exponential growth in the blood of XID mice until the death of the mice at 24 to 36 h. In contrast, PLN reaches a plateau in growth that is maintained for several days. Capsular type 3 and 5 strains exhibited exponential growth and caused rapid death of XID mice following intravenous challenge, similar to the observation with D39. Strains of capsular types 4 and 6 exhibited growth kinetics reminiscent of PLN. Since the observed differences in the pathogenesis of types 3 and 5 compared to 4 and 6 were reminiscent of the effects of pneumolysin deficiency in type 2, we examined the levels of in vitro pneumolysin production for the entire panel of strains. The onset of pneumolysin production in most strains was rapid and occurred near the end of log-phase growth. Differences in in vivo growth patterns of capsular type 2, 3, 4, 5, and 6 strains were not found to be associated with differences in the levels of pneumolysin.     

Changes in serum pepsinogen, gastrin, and immunoglobulin G antibody titers in helicobacter pylori-positive gastric ulcer after eradication of infection

The feasibility of using the spiral nerve cuff electrode design for recordings of respiratory output from the hypoglossal (HG) and phrenic nerves is demonstrated in anesthetized, paralyzed, and artificially ventilated cats. Raw neural discharges of the HG nerve were analyzed in terms of signal-to-noise ratios and frequency spectra. The rectified and integrated moving average activity of the HG nerve had a peak value of 1.74 +/- 0.21 microV and a baseline value of 0.72 +/- 0.11 microV at elevated respiratory drive induced by increases in CO2 or oxygen deprivation when recorded with 10-mm-long cuffs. The frequency content of the HG electroneurogram extended from several hundred hertz to 6 kHz. Spiral nerve cuff recordings without desheathing of the nerve provided large enough signal-to-noise ratios that allowed them to be used as a measure of respiratory output and had much wider frequency bandwidths than the hook electrode preparations. A major advantage of the cuff electrode over the hook electrode was its mechanical stability, which significantly improved the reproducibility of the recordings both in terms of signal amplitudes and frequency contents.     

Toxicity of fumonisin B1 to B6C3F1 mice: a 14-day gavage study

Fumonisin B1 (FB1) is a fungal toxin produced by members of the genus Fusarium. Ingestion of FB1 causes species-specific neurotoxic, nephrotoxic, hepatotoxic and pulmonary effects. The clinical, haematological and pathological responses of adult male and female B6C3F1 mice to FB1 were assessed following 14 daily gavage doses ranging from 1 to 75 mg FB1/kg body weight/day. There were no consistent sex-related changes. Although all responses were modest, the most notable effects of FB1 were on the liver, bone marrow, adrenals and kidneys. In the liver, hepatocellular single cell necrosis, mitosis and anisokaryosis were observed, accompanied by elevated serum ALT. In the kidneys, minor histopathological changes were confined to female mice, while mild decreases in ion transport and increases in blood urea nitrogen were seen only in males. Small changes in glutathione levels were observed in the kidneys and livers of male mice. Adrenal cortical cell vacuolation was observed at 15 mg FB1/kg and higher in females and from 35 mg FB1/kg in males. Serum cholesterol was elevated in both male and female mice, possibly due to FB1-induced changes in lipid metabolism in the liver and adrenals. Although bone marrow cell numbers were unchanged, increases in vacuolated myeloid cells and lymphocytes were observed in female mice. In general, the degree of changes observed indicate that mice are not as sensitive a model of FB1 toxicity as rats.     

Diagnostic implications of kinetics of immunoglobulin M and A antibody responses to Toxoplasma gondii

We evaluated immunoglobulin M (IgM) and IgA assays that could improve the predictive value for recently acquired toxoplasma infection for patients with positive screening test results. Follow-up sera were collected from 82 patients whose initial serum specimen had a reactive anti-Toxoplasma gondii IgM result. According to the evolution of the immune response, patients were divided retrospectively into two groups: one in which a recent infection was unlikely and the other one with an evolving immune response suggestive of recent toxoplasma infection. All IgM and one of three IgA assays used in the study are suitable for screening pregnant patients, with a negative predictive value of 100%. The predictive value of positive results is much lower because of the low prevalence of acute toxoplasmosis in pregnant women and the long persistence of IgM after acute infection. In the present study, all except one IgM enzyme immunoassay remained positive well beyond 6 months after the initial sample was tested. The IgM immunofluorescence test had the shortest persistence of positivity in most cases. IgA tests were either too insensitive or remained reactive too long to be useful for screening pregnant patients. Interpreting enzyme immunoassays with modified cutoff values and the combination of two tests could improve the predictive value of positive results to about 80% in terms of recent infection.     

Antibody responses in serum and lung to intranasal immunization with Haemophilus influenzae type b polysaccharide conjugated to cholera toxin B subunit and tetanus toxoid

AIM: Cholera toxin B subunit (CTB) has previously been used as a mucosal carrier for various vaccine candidate antigens. The objective of this study was to see if coupling a bacterial polysaccharide, Haemophilus influenzae type b capsular polysaccharide (HibCPS), to CTB, either directly or through prior coupling to tetanus toxoid (TT), would improve the immunogenicity of HibCPS after nasal immunization. METHODS: HibCPS was conjugated to CTB, TT or via TT to CTB, using glutaraldehyde or 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDAC) and N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The conjugates were characterized and used for intranasal (IN) and subcutaneous (SC) immunizations of mice. The anti-Hib, -TT and -CTB antibody titers in serum and lungs after the immunizations were measured with ELISA. RESULTS: The HibCTB was poorly immunogenic both given IN and SC compared with HibTT and HibTTCTB, probably because of inefficient coupling. In contrast, the conjugation of CTB to the HibTT conjugate resulted in a preparation which was superior both to the HibTT and the HibCTB conjugates in inducing local IgA and IgG anti-HibCPS antibodies in the lungs. The anti-HibCPS serum IgG titers after IN immunization with the HibTTCTB conjugate were similar to the titers after IN immunization with HibTT, or SC immunization with a commercial HibCRM conjugate vaccine. In contrast to the other conjugates, the HibTTCTB conjugate also gave rise to anti-Hib serum IgA titers. CONCLUSION: We conclude that appropriate conjugation to CTB increases the mucosal immunogenicity of HibCPS, and that intranasal immunization with such a conjugate can give rise to both local and systemic anti-HibCPS antibody responses.     

Development and comparison of assays for measuring immunospecific antibody response in serum of chickens and rabbits immunized with human immunoglobulin G

The antibody response development during polyclonal antibody production is a relevant parameter to monitor during the immunization period to be able to optimize the immunization protocol and to determine the optimal antibody harvest time. Although rabbits and other mammals are most often used for polyclonal antibody production, the chicken is a relevant alternative. There are both scientific reasons, economic reasons, and animal welfare reasons to consider when choosing the chicken instead of a mammal for this purpose, because antibodies in generous quantities can be harvested from the egg yolk. This study compared different assays for measuring antibody response in rabbit and chicken serum. An inhibition liquid phase absorption assay (ILPAA), a rocket immunoelectrophoresis (RIE) assay, and a line immunoelectrophoresis (LIE) assay were compared to ELISAs. The ELISA proved to be the most useful assay for routine use, as it was less time-consuming and because the assay could easily be adapted to both serum antibody types. However, electrophoretic assays were the most useful as combined analytical and quantitative tools and must be considered essential when analyzing specificities of polyclonal antibody preparations.     

IL-12 enhances antibody responses to T-independent polysaccharide vaccines in the absence of T and NK cells

Polysaccharide vaccines to encapsulated bacteria such as Neisseria meningitidis and Streptococcus pneumoniae are weakly immunogenic due to their T-independent (TI) nature. Even when converted to T-dependent forms through conjugation to foreign proteins, polysaccharides induce responses that are deficient in many respects, such as induction of murine IgG2a Ab, the isotype that mediates optimal complement fixation and opsonization. We now show that IL-12 treatment of mice induces significantly increased levels of IgG2a Ab to the model TI-2 Ag, DNP-Ficoll, and to vaccines composed of polysaccharides from pneumococci and meningococci. Use of immunodeficient mice lacking T cells and/or NK cells demonstrated that such cells were not responsible for the observed Ab enhancement. Furthermore, the use of IFN-gamma knockout mice showed that stimulation of TI-2 Ab responses by IL-12 was only partially dependent on IFN-gamma. The ability of IL-12 to dramatically enhance TI Ab responses suggests that IL-12 will be useful as a powerful vaccine adjuvant to induce protective immune responses against encapsulated pathogens.     

Molecular analysis by pulsed-field gel electrophoresis and antibiogram of Streptococcus pneumoniae serotype 6B isolates from selected areas within the United States

Fifty-eight clinical isolates of Streptococcus pneumoniae serotype 6B, including 16 from Alaska, 14 from Arizona, 11 from Washington, and 17 from seven additional states, were analyzed. The antibiograms of these isolates were assigned to 10 antibiotic profiles based on their susceptibilities to penicillin, erythromycin, tetracycline, and trimethoprim-sulfamethoxazole. Thirty-two (55%) of these isolates were penicillin nonsusceptible, while 21 (36%) were intermediate or resistant to three or more antibiotics. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes, and the fragments were resolved by pulsed-field gel electrophoresis (PFGE). The ApaI and SmaI PFGE patterns were combined, and 13 of the 16 Alaskan isolates showed indistinguishable PFGE patterns. One other isolate exhibited highly related ApaI and SmaI PFGE patterns, differing by only one band after restriction with ApaI. Among the 14 isolates from Arizona, 1 was indistinguishable from the predominant ApaI and SmaI PFGE patterns seen in the Alaskan isolates; 5 others were highly related (+/-1 band after cutting with either enzyme) to the Alaskan isolates, suggesting a common ancestral origin. Of the remaining eight isolates, six additional ApaI plus SmaI PFGE patterns were observed. The 28 isolates from the various contiguous states had 22 ApaI plus SmaI PFGE patterns. No correlations were found between specific PFGE patterns, antibiograms, dates of isolation, or geography. The serotype 6B isolates across the contiguous United States were genetically diverse, while the 6B isolates from Alaska appeared to be much less diverse.     

The effect of immunization route on rat serum and tear antibody responses to Chlamydia trachomatis

Using reliable displacement radiobinding assay (RBA) and ELISA, the existence of anti-human growth hormone autoantibodies (hGHAA) was confirmed in idiopathic hypopituitary patients with growth impairment. Six of 35 hypopituitary patients (17.1%) and 1/85 (1.2%) control children proved positive for hGHAA by RBA (>control mean + 3 SD). IgG isotype-hGHAA by ELISAIgG (> control mean + 3 SD) were positive for 6/34 (17.7%) and 3/85 (3.5% hypopituitary and control children, respectively. Due to an asymmetry to the right of the ELISAIgG distribution, an alternative cutoff based on a nonparametric method was obtained, and positive results for hypopituitary children increased to 10/34 (29.4%). Three of 34 hypopituitary patients but no control children were positive for hGHAA of IgM isotype. The hGHAA were detected in children with or without perinatal problems. These autoantibodies may represent markers of a major autoimmune process involving a portion of the anterior pituitary and may contribute to the development of hypopituitarism in over 15% of hypopituitary children.