Characterization and Application of a Glucose-Repressible Promoter in Francisella tularensis

Abstract

Francisella tularensis, the causative agent of tularemia, is a category A biodefense agent. The examination of gene function in this organism is limited due to the lack of available controllable promoters. Here, we identify a promoter element of F. tularensis LVS that is repressed by glucose (termed the Francisella glucose-repressible promoter, or FGRp), allowing the management of downstream gene expression. In bacteria cultured in medium lacking glucose, this promoter induced the expression of a red fluorescent protein allele, tdtomato. FGRp activity was used to produce antisense RNA of iglC, an important virulence factor, which severely reduced IglC protein levels. Cultivation in glucose-containing medium restored IglC levels, indicating the usefulness of this promoter for controlling both exogenous and chromosomal gene expression. Moreover, FGRp was shown to be active during the infection of human macrophages by using the fluorescence reporter. In this environment, the FGRp-mediated expression of antisense iglC by F. tularensis LVS resulted in reduced bacterial fitness, demonstrating the applicability of this promoter. An analysis of the genomic sequence indicated that this promoter region controls a gene, FTL_0580, encoding a hypothetical protein. A deletion analysis determined the critical sites essential for FGRp activity to be located within a 44-bp region. This is the first report of a conditional promoter and the use of antisense constructs in F. tularensis, valuable genetic tools for studying gene function both in vitro and in vivo.

Francisella tularensisΔpyrFMutants Show that Replication in Nonmacrophages Is Sufficient for PathogenesisIn Vivo

“…Buffy coats from blood donations (Central Blood Bank, Pittsburgh, PA) served as the source of human monocytes that were differentiated into macrophages by in vitro culture as has been detailed previously (9,10,27,29,50). For in vitro infections, primary human macrophages were washed and resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% human serum AB (complement replete; GemCell Gemini Bio-Products), 25 mM HEPES, and 1% GlutaMAX and then plated onto Primaria-coated 96-well culture dishes (Becton, Dickinson and Company) at a density of 5.0 ϫ 10 4 cells per well as we have described previously (27).…”

Genetic engineering ofFrancisella tularensisLVS for use as a novel live vaccine platform againstPseudomonas aeruginosainfections

“…This strategy employed the use of a stable Francisella shuttle vector in which the exogenous genes were under the control of either the groE or FGRp promoter. 9,17 These recombinant F. tularensis LVS strains were used to immunize mice to determine if the heterologously-expressed proteins could generate a robust adaptive immune response against P. aeruginosa. Mice that were immunized with recombinant LVS expressing FliC of P. aeruginosa produced a significant level of antibodies specific for P. aeruginosa relative to mock-immunized mice.…”

“…The plasmid pGFLI encoding fliC of P. aeruginosa PA14 under the control of the F. tularensis LVS FGRp promoter 17 was generated as follows. The primers fliCfwdnde and fliCrevbam were used to amplify fliC of P. aeruginosa PA14.…”

“…17 Briefly, F. tularensis LVS bacteria grown on chocolate agar plates were used to inoculate trypticase soy broth supplemented with 0.1% Cysteine HCl (TSBc). This culture was incubated overnight at 37 C with shaking until bacteria reached stationary phase.…”

Working toward the Future: Insights intoFrancisella tularensisPathogenesis and Vaccine Development

“…These vectors can be efficiently transformed into F. tularensis subspecies by electroporation, are stably maintained even in the absence of antibiotic selection, and do not alter virulence characteristics of F. tularensis in vitro or in vivo (124,125,145,146). A variety of pFNLTP1 variants have been generated, and these include derivatives that carry antibiotic resistance elements amenable for use in type A strains of F. tularensis, multiple cloning sites, reporter genes and counterselectable markers, and temperature-sensitive origins of replication (93,125). In addition to their use as complementation and reporter gene platforms, pFNLTP1-based vectors (or vectors that have been derived from them) have been used as delivery vehicles to carry out transposon mutagenesis, targeted allelic exchange, and promoter-trap library construction (22,125,128).…”