title: Development of a gene delivery system in Streptococcus gordonii using thymidylate synthase as a selection marker.
abstract: Streptococcus gordonii, a commensal bacterium of the human oral cavity, is a potential live vaccine vector. In this study, we have developed a system that delivers a vaccine antigen gene onto the chromosome of S. gordonii. The system consisted of a recipient strain, that is a thymidine auxotroph constructed by deletion of a portion of thyA gene, and a linear gene delivery construct, composed of the functional thyA gene, the vaccine antigen gene, and a DNA fragment immediately downstream of thyA. The construct is assembled by a ligation and polymerase chain reaction strategy. Upon introduction into the thyA mutant, the vaccine antigen gene integrated into the chromosome via a double crossing-over event. Using the above strategy, a test vaccine antigen gene coding for a fusion protein composed of the Bordetella pertussis filamentous hemagglutinin type I domain and the single chain antibody against complement receptor 1 was successfully delivered to S. gordonii. The resulting S. gordonii expressed the fusion protein and the delivered gene was stable in the bacterium in vitro and in a mouse colonization experiment. Mice colonized by the fusion protein-expressing S. gordonii developed antibodies that recognized the native filamentous hemagglutinin protein suggesting that an immune response was elicited.
authors.affiliation: Department of Microbiology and Immunology, Dalhousie University, Halifax, NS B3H 1X5, Canada; Canadian Center for Vaccinology, Dalhousie University and the IWK Health Centre, Halifax, NS B3K 6R8, Canada; Department of Pediatrics, Faculty of Medicine, Dalhousie University and the IWK Health Centre, Halifax, NS B3K 6R8, Canada; Department of Applied Oral Sciences, Faculty of Dentistry, Dalhousie University, Halifax, NS B3H 4R2, Canada. Electronic address: Song.Lee@Dal.Ca.
authors.forename: Song F
authors.initials: SF
authors.lastname: Lee
authors.affiliation: Department of Microbiology and Immunology, Dalhousie University, Halifax, NS B3H 1X5, Canada; Canadian Center for Vaccinology, Dalhousie University and the IWK Health Centre, Halifax, NS B3K 6R8, Canada.
authors.forename: Maram
authors.initials: M
authors.lastname: Hulbah
authors.affiliation: Department of Microbiology and Immunology, Dalhousie University, Halifax, NS B3H 1X5, Canada; Canadian Center for Vaccinology, Dalhousie University and the IWK Health Centre, Halifax, NS B3K 6R8, Canada; Department of Pediatrics, Faculty of Medicine, Dalhousie University and the IWK Health Centre, Halifax, NS B3K 6R8, Canada.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Halperin
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics / Q000276:immunology; D000818:Animals; D000907:Antibodies, Bacterial; D002876:Chromosomes, Bacterial / Q000235:genetics; D018014:Gene Transfer Techniques; D006801:Humans; D000071497:Immunogenicity, Vaccine; D051379:Mice; D009055:Mouth / Q000382:microbiology; D009154:Mutation; D011951:Receptors, Complement / Q000276:immunology; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies / Q000235:genetics; D054773:Streptococcus gordonii / Q000201:enzymology* / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D013936:Thymidine / Q000235:genetics; D013940:Thymidylate Synthase / Q000235:genetics*; D014613:Vaccines, Attenuated / Q000737:chemistry / Q000235:genetics; D010566:Virulence Factors, Bordetella / Q000235:genetics / Q000276:immunology
chemical_list: D018829:Adhesins, Bacterial; D000907:Antibodies, Bacterial; D011951:Receptors, Complement; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D014613:Vaccines, Attenuated; D010566:Virulence Factors, Bordetella; C065273:filamentous hemagglutinin adhesin, Bordetella pertussis; D013940:Thymidylate Synthase; D013936:Thymidine
keywords: Gene delivery; Pertussis; Streptococcus gordonii; thyA