Method and parameters for genetic transformation of Streptococcus sanguis challis

Pozzi, Gianni; Musmanno, R. A.; Lievens, Patricia; Oggioni, Marco R.; Plevani, Paolo; Manganelli, Riccardo

doi:10.1016/0923-2508(90)90060-4

Cited by 63 publications

(49 citation statements)

References 46 publications

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“…Plasmid pSMB120 was then used to transform competent cells of S. gordonii GP1221.1 to obtain the recombinant strain GP1246. Procedures for cloning gene fusions in E. coli, transformation of S. gordonii, and scoring and genetic analysis of transformants were as previously described (37)(38)(39).…”

Section: Methodsmentioning

confidence: 99%

Immunogenicity of the B Monomer ofEscherichia coliHeat-Labile Toxin Expressed on the Surface ofStreptococcus gordonii

et al. 2000

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The B monomer of the Escherichia coli heat-labile toxin (LTB) was expressed on the surface of the human oral commensal bacterium Streptococcus gordonii. Recombinant bacteria expressing LTB were used to immunize BALB/c mice subcutaneously and intragastrically. The LTB monomer expressed on the streptococcal surface proved to be highly immunogenic, as LTB-specific immunoglobulin G (IgG) serum titers of 140,000 were induced after systemic immunization. Most significantly, these antibodies were capable of neutralizing the enterotoxin in a cell neutralization assay. Following mucosal delivery, antigen-specific IgA antibodies were found in feces and antigen-specific IgG antibodies were found in sera. Analysis of serum IgG subclasses showed a clear predominance of IgG1 when recombinant bacteria were inoculated subcutaneously, while a prevalence of IgG2a was observed upon intragastric delivery, suggesting, in this case, the recruitment of a Th1 type of immune response.

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Section: Methodsmentioning

confidence: 99%

Immunogenicity of the B Monomer ofEscherichia coliHeat-Labile Toxin Expressed on the Surface ofStreptococcus gordonii

et al. 2000

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“…Plasmids were purified using a Qiagen plasmid midi kit (Qiagen GmbH, Hilden, Germany). Genetic transformation of S. gordonii Challis was performed as described elsewhere (39). PCR amplification was carried out with a GeneAmp PCR system 9700 (Perkin-Elmer, Norwalk, Conn.).…”

Section: Methodsmentioning

confidence: 99%

“…DNA was prepared by published methods (30,39). Plasmids were purified using a Qiagen plasmid midi kit (Qiagen GmbH, Hilden, Germany).…”

Section: Methodsmentioning

confidence: 99%

Study of Staphylococcus aureus Pathogenic Genes by Transfer and Expression in the Less Virulent Organism Streptococcus gordonii

et al. 2001

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Because Staphylococcus aureus strains contain multiple virulence factors, studying their pathogenic role by single-gene inactivation generated equivocal results. To circumvent this problem, we have expressed specific S. aureus genes in the less virulent organism Streptococcus gordonii and tested the recombinants for a gain of function both in vitro and in vivo. Clumping factor A (ClfA) and coagulase were investigated. Both gene products were expressed functionally and with similar kinetics during growth by streptococci and staphylococci. ClfA-positive S. gordonii was more adherent to platelet-fibrin clots mimicking cardiac vegetations in vitro and more infective in rats with experimental endocarditis (P < 0.05). Moreover, deleting clfA from clfA-positive streptococcal transformants restored both the low in vitro adherence and the low in vivo infectivity of the parent. Coagulase-positive transformants, on the other hand, were neither more adherent nor more infective than the parent. Furthermore, coagulase did not increase the pathogenicity of clfA-positive streptococci when both clfA and coa genes were simultaneously expressed in an artificial minioperon in streptococci. These results definitively attribute a role for ClfA, but not coagulase, in S. aureus endovascular infections. This gain-offunction strategy might help solve the role of individual factors in the complex the S. aureus-host relationship.

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“…The wild-type S. gordonii strain GP204 (29) and the OVA-expressing recombinant GP1252 were grown at 37°C in tryptic soy broth without dextrose (Difco) and harvested by centrifugation at the end of the exponential phase of growth. Bacterial cells were then washed and resuspended in fresh medium containing 10% glycerol at 1:500 of the original culture volume.…”

Section: Methodsmentioning

confidence: 99%

Bacteria-induced neo-biosynthesis, stabilization, and surface expression of functional class I molecules in mouse dendritic cells

Rescigno

Citterio

Théry

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

227

188

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Here, we show that bacteria induce de novo synthesis of both major histocompatability complex (MHC) class I and II molecules in a mouse dendritic cell culture system. The neo-biosynthesis of MHC class I molecules is delayed as compared with that of MHC class II. Furthermore, bacteria stabilize MHC class I molecules by a 3-fold increase of their half-life. This has important consequences for the capacity of dendritic cells to present bacterial antigens in the draining lymph nodes. In addition, a model antigen, ovalbumin, expressed on the surface of recombinant Streptococcus gordonii is processed and presented on MHC class I molecules. This presentation is 10 6 times more efficient than that of soluble OVA protein. This exogenous pathway of MHC class I presentation is transporter associated with antigen processing (TAP)-dependent, indicating that there is a transport from phagolysosome to cytosol in dendritic cells. Thus, bacteria are shown to be a potentially useful mean for the correct delivery of exogenous antigens to be presented efficiently on MHC class I molecules.

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Method and parameters for genetic transformation of Streptococcus sanguis challis

Cited by 63 publications

References 46 publications

Immunogenicity of the B Monomer ofEscherichia coliHeat-Labile Toxin Expressed on the Surface ofStreptococcus gordonii

Immunogenicity of the B Monomer ofEscherichia coliHeat-Labile Toxin Expressed on the Surface ofStreptococcus gordonii

Study of Staphylococcus aureus Pathogenic Genes by Transfer and Expression in the Less Virulent Organism Streptococcus gordonii

Bacteria-induced neo-biosynthesis, stabilization, and surface expression of functional class I molecules in mouse dendritic cells

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