scholarly journals Construction of Otherwise Isogenic Serotype 6B, 7F, 14, and 19F Capsular Variants of Streptococcus pneumoniae Strain TIGR4

2003 ◽  
Vol 69 (12) ◽  
pp. 7364-7370 ◽  
Author(s):  
Krzysztof Trzcinski ◽  
Claudette M. Thompson ◽  
Marc Lipsitch
Keyword(s):  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Rflp Analysis ◽  
Capsular Type ◽  
Chromosomal Dna ◽  
Subsequent Transformation ◽  
Polysaccharide Synthesis ◽  
Quellung Reaction ◽  
Flanking Regions

ABSTRACT The polysaccharide capsule is the primary virulence factor in Streptococcus pneumoniae. There are at least 90 serotypes of S. pneumoniae, identified based on the immunogenicity of different capsular sugars. The aim of this study was to construct pneumococcal strains that are isogenic except for capsular type. Serotype 4 strain TIGR4 was rendered unencapsulated by recombinational replacement of the capsular polysaccharide synthesis (cps) locus with the bicistronic Janus cassette (C. K. Sung, J. P. Claverys, and D. A. Morrison, Appl. Environ. Microbiol. 67:5190-5196, 2001). In subsequent transformation with chromosomal DNA, the cassette was replaced by the cps locus derived from a strain of a different serotype, either 6B, 7F, 14, or 19F. To minimize the risk of uncontrolled recombinational replacements in loci other than cps, the TIGRcps::Janus strain was“ backcross” transformed three times with chromosomal DNA of subsequently constructed capsular type transformants. Capsular serotypes were confirmed in all new capsule variants by the Quellung reaction. Restriction fragment length polymorphism (RFLP) analysis of the cps locus confirmed the integrity of the cps region transformed into the TIGR strain, and RFLP of the flanking regions confirmed their identities with the corresponding regions of the recipient. Transformants had in vitro growth rates greater than or equal to that of TIGR4. All four strains were able to colonize C57BL/6 mice (female, 6 weeks old) for at least 7 days when mice were intranasally inoculated with 6 × 106 to 8 × 106 CFU. The constructed capsular variants of TIGR4 are suitable for use in studies on the role of S. pneumoniae capsular polysaccharide in immunity, colonization, and pathogenesis.

scholarly journals THE PRODUCTION OF MONOCLONAL ANTIBODIES TO TETRASACCHARIDE - SYNTHETIC ANALOGUE OF THE CAPSULAR POLYSACCHARIDE OF STREPTOCOCCUS PNEUMONIAE OF SEROTYPE 14 AND THEIR IMMUNOCHEMICAL CHARACTERIZATION

2018 ◽  
pp. 26-31
Author(s):  
I. V. Yakovleva ◽  
E. A. Kurbatova ◽  
E. A. Akhmatova ◽  
E. V. Sukhova ◽  
D. V. Yashunsky ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Solid Phase ◽  
Synthetic Analogue ◽  
Immunochemical Characterization ◽  
Carbohydrate Epitopes ◽  
First Time

Aim. Production of monoclonal antibodies (mAb) to synthetic tetrasaccharide - repeating unit of the capsular polysaccharide (CP) of Streptococcus pneumoniae serotype 14 and their immunochemical characterization. Materials and methods. In order to generate the hybridoma producing mAb, mice were immunized with synthetic tetrasaccharide conjugated with bovine serum albumin (BSA) with following hybridization of B lymphocytes with mouse myeloma cells. Antibodies were obtained in vitro andin vivo. Immunochemical characterization of mAb to tetrasaccharide was carried out using a variety of ELISA options. Results. For the first time obtained mouse hybridoma, producing IgM to tetrasacchride. The IgM titer of anti-tetrasacharide antibodies in supernatants of clones and in the ascitic fluid of mice in ELISA detected by biotinylated tetrasaccharide and synthetic CP adsorbed on the solid phase was higher compared to the use of bacterial CP as well cover antigen. In the reaction of inhibition of the ELISA, the mAb recognized the corresponding carbohydrate epitopes of the bacterial CP of S. pneumoniae serotype 14 dissolved in the liquid phase better than tetrasaccharide ligand and synthetic CP. Conclusion. To detect mAb to tetrasaccharide in ELISA preferably to use synthetic analogues of the CP as solid phase antigens. The obtained mAb to tetrasaccharide can be used to determine the representation of the protective tetrasaccharide epitope of CP in the development of pneumococcal vaccines.

Presence of Two Plasminogen Alleles in Normal Populations

1998 ◽  
Vol 79 (01) ◽  
pp. 150-154 ◽  
Author(s):  
Masafumi Kida ◽  
Masuyo H.- Kawabata ◽  
Tomio Yamazaki ◽  
Akitada Ichinose
Keyword(s):  
Rflp Analysis ◽  
Normal Subjects ◽  
Nucleotide Sequences ◽  
Normal Populations ◽  
Pcr Rflp ◽  
Expression Activity ◽  
Flanking Regions

SummaryWhen we compared nucleotide sequences of the 5’-flanking regions for plasminogen genes from 11 individuals, six substitutions were identified even among normal subjects. A new haplotype (termed allele II) was screened by PCR-RFLP analysis among 54 Japanese and 58 Caucasian normal subjects. The frequency of allele II was 0.787 in the Japanese and 0.560 in the Caucasians, indicating that the ratio of alleles differs between populations. Examination of 118 cases with dysplasminogenemia revealed that the Ala601-Thr mutation was present on allele I in most cases. This mutation was also associated with allele II in one-fourth of all cases, suggesting possible recombination within the plasminogen gene. Interestingly, we previously demonstrated that the expression activity of allele II was about 1.8 fold that of allele I in vitro.

scholarly journals Discovery of a Streptococcus pneumoniae serotype 33F capsular polysaccharide locus that lacks wcjE and contains a wcyO pseudogene

2018 ◽  
Author(s):  
Sam Manna ◽  
Eileen M. Dunne ◽  
Belinda D. Ortika ◽  
Casey L. Pell ◽  
Mike Kama ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Sequence Similarity ◽  
Phylogenetic Analyses ◽  
Latex Agglutination ◽  
Reference Sequence ◽  
Middle Income ◽  
Capsule Locus ◽  
Vaccine Impact ◽  
Quellung Reaction

AbstractObjectivesAs part of large on-going vaccine impact studies in Fiji and Mongolia, we identified 25/2750 (0.9%) of nasopharyngeal swabs by microarray that were positive for Streptococcus pneumoniae contained pneumococci with a divergent 33F capsular polysaccharide locus (designated ‘33F-1’). We investigated the 33F-1 capsular polysaccharide locus to better understand the genetic variation and its potential impact on serotyping results.MethodsWhole genome sequencing was conducted on ten 33F-1 pneumococcal isolates. Initially, sequence reads were used for molecular serotyping by PneumoCaT. Phenotypic typing of 33F-1 isolates was then performed using the Quellung reaction and latex agglutination. Genome assemblies were used in phylogenetic analyses of each gene in the capsular locus to investigate genetic divergence.ResultsAll ten pneumococcal isolates with the 33F-1 cps locus typed as 33F by Quellung and latex agglutination. Unlike the reference 33F capsule locus sequence, DNA microarray and PneumoCaT analyses found that 33F-1 pneumococci lack the wcjE gene, and instead contain wcyO with a frameshift mutation. Phylogenetic analyses found the wzg, wzh, wzd, wze, wchA, wciG and glf genes in the 33F-1 cps locus had higher DNA sequence similarity to homologues from other serotypes than to the 33F reference sequence.ConclusionsWe have discovered a novel genetic variant of serotype 33F, which lacks wcjE and contains a wcyO pseudogene. This finding adds to the understanding of molecular epidemiology of pneumococcal serotype diversity, which is poorly understood in low and middle-income countries.

scholarly journals Single-Step Capsular Transformation and Acquisition of Penicillin Resistance in Streptococcus pneumoniae

2004 ◽  
Vol 186 (11) ◽  
pp. 3447-3452 ◽  
Author(s):  
Krzysztof Trzciński ◽  
Claudette M. Thompson ◽  
Marc Lipsitch
Keyword(s):  
Streptococcus Pneumoniae ◽  
Multidrug Resistant ◽  
Single Step ◽  
Susceptible Strain ◽  
Polymorphism Analysis ◽  
Capsule Locus ◽  
Elevated Rate ◽  
Quellung Reaction ◽  
Natural Settings ◽  
Flanking Regions

ABSTRACT The capsule (cps) locus of Streptococcus pneumoniae is flanked by the pbp2x and pbp1a genes, coding for penicillin-binding proteins, enzymes involved in cell wall synthesis that are targets for β-lactams. This linkage suggested to us that selection for β-lactam resistance might coselect for capsular transformants. The recombination event would then involve PBP genes, as well as the cps operon, and would change both the serotype and the resistance profile of the strain. We transformed β-lactam-susceptible strain TIGR4 by using whole genomic DNA extracted from multidrug-resistant strain GA71, a serotype 19F variant of pneumococcal clone Spain23F-1, and selected β-lactam-resistant transformants. Smooth colonies appearing on selective plates were subcultured, serotyped by the Quellung reaction, and genotyped to confirm the presence of the GA71 pbp2x-cps19-pbp1a locus in the TIGR4 genetic background by restriction fragment length polymorphism analysis of the whole locus and its flanking regions. The results showed that a new serotype, combined with resistance to β-lactams, could emerge in a susceptible strain via a single transformation event. Quantitative analysis showed that transfer of the cps locus had occurred at an elevated rate in β-lactam-selected transformants. This suggests that in natural settings selection by host immunity and selection by antibiotics may be interrelated because of “hitchhiking” effects due to linkage of resistance determinants and the capsule locus.

scholarly journals Type III Group B Streptococcal Polysaccharide Induces Antibodies That Cross-React with Streptococcus pneumoniae Type 14

2002 ◽  
Vol 70 (4) ◽  
pp. 1724-1738 ◽  
Author(s):  
Hilde-Kari Guttormsen ◽  
Carol J. Baker ◽  
Moon H. Nahm ◽  
Lawrence C. Paoletti ◽  
Susu M. Zughaier ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Bacterial Infections ◽  
Capsular Polysaccharide ◽  
Sialic Acid Residue ◽  
Coating Antigen ◽  
Type Iii ◽  
Young Infants ◽  
Covalently Linked ◽  
Group B

ABSTRACT Covalent linkage of a bacterial polysaccharide to a protein greatly enhances the carbohydrate's immunogenicity and its binding to solid surfaces in immunoassays. These findings have spurred the development of glycoconjugate vaccines to prevent serious bacterial infections as well as the use of glycoconjugates as coating antigens in bioassays. We evaluated sera from women immunized with unconjugated group B streptococcal (GBS) type III (GBS III) polysaccharide (IIIPS) or with IIIPS covalently linked to tetanus toxoid to assess specificity, sensitivity, and parallelism in dilution curves in two GBS III enzyme-linked immunosorbent assays (ELISAs). One assay used IIIPS mixed with methylated human serum albumin (IIIPS + mHSA) as the coating antigen, and the other used IIIPS covalently linked to HSA (III-HSA). Each coating antigen was associated with a highly specific GBS III bioassay. The sensitivity was higher in the III-HSA ELISA, in which conjugated IIIPS is bound to the plates. Parallelism in titration curves was observed in the III-HSA but not in the IIIPS + mHSA ELISA. The excellent correlation between the concentrations of GBS IIIPS-specific immunoglobulin G (IgG) and the opsonophagocytic activity of these antibodies indicated that the III-HSA assay can predict functionality of vaccine-induced IgG against GBS III disease. The structure of the repeating unit of the capsular polysaccharide of GBS III differs from that of Streptococcus pneumoniae type 14 (Pn14 PS) only by the presence on GBS III of a sialic acid residue at the end of the side chain. The majority of healthy adults responding to GBS III vaccines with a fourfold or greater increase in GBS III-specific IgG antibodies developed antibodies cross-reacting with Pn14 PS (i.e., desialylated GBS IIIPS). The proportion of GBS vaccine responders who developed IgG to the desialylated IIIPS did not depend on whether IIIPS was given in the unconjugated or conjugated form. When present, these vaccine-induced cross-reacting antibodies conferred in vitro antibody-mediated opsonophagocytosis and killing of both GBS III and Pn14, two pathogens that cause invasive disease in young infants.

scholarly journals Efficacy of Opsonic and Nonopsonic Serotype 3 Pneumococcal Capsular Polysaccharide-Specific Monoclonal Antibodies against Intranasal Challenge with Streptococcus pneumoniae in Mice

2009 ◽  
Vol 77 (4) ◽  
pp. 1502-1513 ◽  
Author(s):  
Haijun Tian ◽  
Sarah Weber ◽  
Peter Thorkildson ◽  
Thomas R. Kozel ◽  
Liise-anne Pirofski
Keyword(s):  
Monoclonal Antibodies ◽  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Passive Immunization ◽  
Immunodeficient Mice ◽  
Pneumococcal Strain ◽  
J774 Cells ◽  
Complement Component 3

ABSTRACT Serotype-specific antibodies to pneumococcal capsular polysaccharide (PPS) are a critical component of vaccine-mediated immunity to Streptococcus pneumoniae. In this study, we investigated the in vitro opsonophagocytic activities of three PPS-specific mouse immunoglobulin G1 monoclonal antibodies (MAbs), 1E2, 5F6, and 7A9, and determined their in vivo efficacies against intranasal challenge with WU2, a serotype 3 pneumococcal strain, in normal and immunodeficient mice. The MAbs had different in vitro activities in a pneumococcal killing assay: 7A9 enhanced killing by mouse neutrophils and J774 cells in the presence of a complement source, whereas 5F6 promoted killing in the absence, but not the presence, of complement, and 1E2 did not promote killing under any conditions. Nonetheless, all three MAbs protected normal and complement component 3-deficient mice from a lethal intranasal challenge with WU2 in passive-immunization experiments in which 10 μg of the MAbs were administered intraperitoneally before intranasal challenge. In contrast, only 1E2 protected Fcγ receptor IIB knockout (FcγRIIB KO) mice and mice that were depleted of neutrophils with the MAb RB6, whereas 7A9 and 5F6 required neutrophils and FcγRIIB to mediate protection. Conversely, 7A9 and 5F6 protected FcγR KO mice, but 1E2 did not. Hence, the efficacy of 1E2 required an activating FcγR(s), whereas 5F6 and 7A9 required the inhibitory FcγR (FcγRIIB). Taken together, our data demonstrate that both MAbs that do and do not promote pneumococcal killing in vitro can mediate protection in vivo, although their efficacies depend on different host receptors and/or components.

scholarly journals A Capsular Polysaccharide-Specific Antibody Alters Streptococcus pneumoniae Gene Expression during Nasopharyngeal Colonization of Mice

2018 ◽  
Vol 86 (7) ◽  
Author(s):  
Christopher R. Doyle ◽  
Jee-Young Moon ◽  
Johanna P. Daily ◽  
Tao Wang ◽  
Liise-anne Pirofski
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Vaccine Development ◽  
Lavage Fluid ◽  
Uptake System ◽  
Content Type

ABSTRACT Pneumococcal conjugate vaccines (PCV) elicit opsonophagocytic (opsonic) antibodies to pneumococcal capsular polysaccharides (PPS) and reduce nasopharyngeal (NP) colonization by vaccine-included Streptococcus pneumoniae serotypes. However, nonopsonic antibodies may also be important for protection against pneumococcal disease. For example, 1E2, a mouse IgG1 monoclonal antibody (MAb) to the serotype 3 (ST3) PPS (PPS3), reduced ST3 NP colonization in mice and altered ST3 gene expression in vitro . Here, we determined whether 1E2 affects ST3 gene expression in vivo during colonization of mice by performing RNA sequencing on NP lavage fluid from ST3-infected mice treated with 1E2, a control MAb, or phosphate-buffered saline. Compared to the results for the controls, 1E2 significantly altered the expression of over 50 genes. It increased the expression of the piuBCDA operon, which encodes an iron uptake system, and decreased the expression of dpr , which encodes a protein critical for resistance to oxidative stress. 1E2-mediated effects on ST3 in vivo required divalent binding, as Fab fragments did not reduce NP colonization or alter ST3 gene expression. In vitro , 1E2 induced dose-dependent ST3 growth arrest and altered piuB and dpr expression, whereas an opsonic PPS3 MAb, 5F6, did not. 1E2-treated bacteria were more sensitive to hydrogen peroxide and the iron-requiring antibiotic streptonigrin, suggesting that 1E2 may increase iron import and enhance sensitivity to oxidative stress. Finally, 1E2 also induced rapid capsule shedding in vitro , suggesting that this may initiate 1E2-induced changes in sensitivity to oxidative stress and gene expression. Our data reveal a novel mechanism of direct, antibody-mediated antibacterial activity that could inform new directions in antipneumococcal therapy and vaccine development.

scholarly journals Targeting Streptococcus pneumoniae UDP-glucose pyrophosphorylase (UGPase): in vitro validation of a putative inhibitor

2020 ◽  
Vol 14 (1) ◽  
pp. 26-33
Author(s):  
Monica Sharma ◽  
Swati Sharma ◽  
Pallab Ray ◽  
Anuradha Chakraborti
Keyword(s):  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Inhibitory Concentration ◽  
A549 Cells ◽  
Host Cells ◽  
Genome Plasticity ◽  
Leloir Pathway ◽  
Tnf Α ◽  
Respiratory Epithelial

Background: Genome plasticity of Streptococcus pneumoniae is responsible for the reduced efficacy of various antibiotics and capsular polysaccharide based vaccines. Therefore targets independent of capsular types are sought to control the pneumococcal pathogenicity. UcrDP-glucose pyrophosphorylase (UGPase) is one such desired candidate being responsible for the synthesis of UDP-glucose, a sugar-precursor in capsular biosynthesis and metabolic Leloir pathway. Being crucial to pneumococcal pathobiology, the effect of UGPase inhibition on virulence was evaluated in vitro. Methods: A putative inhibitor (UDP) was evaluated for effective inhibitory concentration in S. pneumoniae and A549 cells, its efficacy and toxicity. Effect of UDP on adherence and phagocytosis was measured in human respiratory epithelial (A549 and HEp-2) and macrophage (THP1 and J774.A.1) cell lines respectively. Results: A differential effective inhibitory concentration of UDP for UGPase inhibition was observed in S. pneumoniae and A549 cells i.e. 5 µM and 100 µM respectively. UDP treatments lowered percent cytotoxicity in pneumococcal infected monolayers and didn't exert adverse effects on viabilities. S. pneumoniae adherence to host cells was decreased significantly with UDP treatments. UDP induced the secretion of IL-1β, TNF-α, IL-6, and IL-8 and increased pneumococcal phagocytosis. Conclusion: Our study shows UDP mediated decrease in the virulence of S. pneumoniae and demonstrates UDP as an effective inhibitor of pneumococcal UGPase.

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