Genetic tools for highly pathogenic Francisella tularensis subsp. tularensis

Abstract

This paper is the first detailed description of the development and use of new genetic tools specifically for the safe manipulation of highly pathogenic Francisella tularensis subsp. tularensis. Most of these tools are also demonstrated to work with other F. tularensis subspecies. Kanamycin and hygromycin resistance determinants that function as genetic markers in F. tularensis subsp. tularensis strain Schu and sets of episomal shuttle vectors that are either unstable or stably maintained in the absence of selection were developed. In addition, the hyg gene, expressed from the F. tularensis groESL promoter, was successfully used as a marker for transposon mutagenesis. This work also includes the development of sacB-based suicide plasmids expressing kanamycin resistance that can be used for electroporation-mediated allelic exchange of unmarked mutations in Schu and the F. tularensis live vaccine strain (LVS). Using these plasmids, the two predicted b-lactamase genes, blaA and blaB, in Schu and LVS were deleted. Only the DblaB1 mutants had increased susceptibility to ampicillin, and this phenotype was complemented by a plasmid expressing blaB + . The results suggest that the b-lactam antibiotic resistance phenotype of Schu and LVS is likely due to only one of the two b-lactamase genes present and that ampicillin resistance can be used as an additional selectable marker in b-lactamase deletion mutants. The collection of tools presented in this report will be helpful for the genetic analyses of F. tularensis subsp. tularensis pathogenesis.

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

“…Genetic tools have been developed that could allow for stable, safe, and effective vaccine strains. [26][27][28][29][30] However, viral proteins requiring glycosylation by eukaryotic host machinery may not be compatible for use with this system. Nevertheless, there is immense potential for F. tularensis LVS to express heterologous bacterial toxoids, surface proteins, and enzymes for lipid or carbohydrate biosynthetic pathways to direct the immune response against the cognate pathogens.…”

“…Future studies should focus on utilizing existing molecular tools to generate stable recombinant F. tularensis LVS bacteria encoding chromosomal copies of selected heterologous genes. [26][27][28][29][30] Aside from adjusting expression levels by F. tularensis LVS, more robust immune responses may be attained by altering the route of immunization or utilizing a boost following vaccination. 31 It is also possible that lack of pre-protein processing may have altered the antigenicity of the recombinant proteins.…”

Development of novel plasmid vectors and a promoter trap system in Francisella tularensis compatible with the pFLN10 based plasmids

“…1). The literature in the last year has contained descriptions of a new transposon mutagenesis system for F. tularensis as well as the description of the plasmids available for the use in F. tularensis (Lovullo et al, 2006), however, both were variations of the existing technologies. To date the known shuttle vectors for use in F. tularensis and Escherichia coli are all based on the F. novicida cryptic plasmid pFNL10 (Lovullo et al, 2006;Maier et al, 2004;Norqvist et al, 1996;Pavlov et al, 1996).…”

“…In this work, we describe the construction of two plasmid vectors for use in F. tularensis that are not based on the pFNL10 plasmid and thus can work in concert with these established F. tularensis vectors for complementation and or multiple gene replacements (Lovullo et al, 2006;Maier et al, 2004;Norqvist et al, 1996;Pavlov et al, 1996). We have constructed three novel plasmids named pCU18, pCUG1, and pCUG2.…”

Single-copy chromosomal integration systems for Francisella tularensis

“…These include Escherichia coli-Francisella shuttle vectors (Bina et al, 2006;LoVullo et al, 2006;Ludu et al, 2008;Maier et al, 2004;Rasko et al, 2007), random transposon mutagenesis systems based on EZ-Tn5, Himar1 and Tn5 (Buchan et al, 2008;Kawula et al, 2004;LoVullo et al, 2006;Maier et al, 2006;Qin & Mann, 2006), as well as methods for allelic exchange (Golovliov et al, 2003;LoVullo et al, 2006;Ludu et al, 2008;Rodriguez et al, 2008;Twine et al, 2005). One methodology that has not been fully explored is the integration of genetic elements into the F. tularensis chromosome.…”