title: A stable luciferase reporter plasmid for in vivo imaging in murine models of Staphylococcus aureus infections.
abstract: In vivo imaging of bioluminescent bacteria permits their visualization in infected mice, allowing spatial and temporal evaluation of infection progression. Most available bioluminescent strains were obtained by integration of the luciferase genes into the bacterial chromosome, a challenging and time-consuming approach. Recently, episomal plasmids were used, which were introduced in bacteria and expressed all genes required for bioluminescence emission. However, the plasmid was progressively lost in vitro and in vivo, if bacteria were not maintained under antibiotic selective pressure. Increased stability could be obtained inserting into the plasmid backbone sequences that assured plasmid partition between daughter bacterial cells, or caused death of bacteria that had lost the plasmid. So far, no detailed analysis was performed of either plasmid stability in vivo or contribution of different stabilizing sequence types. Here we report the construction of a plasmid, which includes the Photorhabdus luminescens lux cassette expressed under the control of a Staphylococcus aureus specific gene promoter, and toxin/antitoxin (T/A) and partition sequences (Par) conferring stability and transmissibility of the plasmid. Following infection of mice with S. aureus carrying this plasmid, we demonstrated that the promoter-lux fusion was functional in vivo, that the plasmid was retained by 70-100% of bacterial cells 7 days post-infection, and that both stabilizing sequence types were required to maximize plasmid retention. These data suggest that the plasmid can be a valuable tool to study gene expression and bacterial spread in small laboratory animals infected with S. aureus or possibly other Gram-positive human pathogens.
authors.affiliation: , GSK Vaccines S.r.l., Via Fiorentina 1, 53100, Siena, Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Bacconi
authors.affiliation: , GSK Vaccines S.r.l., Via Fiorentina 1, 53100, Siena, Italy.
authors.forename: Andreas F
authors.initials: AF
authors.lastname: Haag
authors.affiliation: , GSK Vaccines S.r.l., Via Fiorentina 1, 53100, Siena, Italy.
authors.forename: Antonina
authors.initials: A
authors.lastname: Torre
authors.affiliation: , GSK Vaccines S.r.l., Via Fiorentina 1, 53100, Siena, Italy.|Department Pharmacy and Biotechnology, University of Bologna, Via Belmeloro 6, 40126, Bologna, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Castagnetti
authors.affiliation: , GSK Vaccines S.r.l., Via Fiorentina 1, 53100, Siena, Italy.
authors.forename: Emiliano
authors.initials: E
authors.lastname: Chiarot
authors.affiliation: , GSK Vaccines S.r.l., Via Fiorentina 1, 53100, Siena, Italy.
authors.forename: Isabel
authors.initials: I
authors.lastname: Delany
authors.affiliation: , GSK Vaccines S.r.l., Via Fiorentina 1, 53100, Siena, Italy. giuliano.x.bensi@gsk.com.
authors.forename: Giuliano
authors.initials: G
authors.lastname: Bensi
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism; D003952:Diagnostic Imaging / Q000379:methods*; D004195:Disease Models, Animal; D005260:Female; D017930:Genes, Reporter; D005818:Genetic Engineering; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D008163:Luminescent Measurements; D051379:Mice; D020637:Photorhabdus / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D013203:Staphylococcal Infections / Q000000981:diagnostic imaging* / Q000382:microbiology; D013211:Staphylococcus aureus / Q000235:genetics* / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D006460:Hemolysin Proteins; C021091:staphylococcal alpha-toxin; D008156:Luciferases
keywords: Bioluminescence; In vivo imaging; Plasmid; Staphylococcus aureus; Toxin/antitoxin