Generation and Characterization of an Attenuated Mutant in a Response Regulator Gene of Francisella tularensis Live Vaccine Strain (LVS)

Francisella tularensis is a zoonotic bacterium that must exist in diverse environments ranging from arthropod vectors to mammalian hosts. To better understand how virulence genes are regulated in these different environments, a transcriptional response regulator gene (genome locus FTL0552) was deleted in F. tularensis live vaccine strain (LVS). The FTL0552 deletion mutant exhibited slightly reduced rates of extracellular growth but was unable to replicate or survive in mouse macrophages and was avirulent in the mouse model using either BALB/c or C57BL/6 mice. Mice infected with the FTL0552 mutant produced reduced levels of inflammatory cytokines, exhibited reduced histopathology, and cleared the bacteria quicker than mice infected with LVS. Mice that survived infection with the FTL0552 mutant were afforded partial protection when challenged with a lethal dose of the virulent SchuS4 strain (4 of 10 survivors, day 21 postinfection) when compared to naive mice (0 of 10 survivors by day 7 postinfection). Microarray experiments indicate that 148 genes are regulated by FTL0552. Most of the genes are downregulated, indicating that FTL0552 controls transcription of genes in a positive manner. Genes regulated by FTL0552 include genes located within the Francisella pathogenicity island that are essential for intracellular survival and virulence of F. tularensis. Further, a mutant in FTL0552 or the comparable locus in SchuS4 (FTT1557c) may be an alternative candidate vaccine for tularemia

Preliminary analysis and annotation of the partial genome sequence of Francisella tularensis strain Schu 4.

Francisella tularensis, the aetiological agent of tularemia, is an important pathogen throughout much of the Northern hemisphere. We have carried out sample sequencing of its genome in order to gain a greater insight into this organism about which very little is known, especially at the genetic level. Nucleotide sequence data from a genomic DNA shotgun library of the virulent F. tularensis strain Schu 4 has been partially assembled to provide 1.83 Mb of the genome sequence. A preliminary analysis of the F. tularensis genome sequence has been performed and the data compared with 20 fully sequenced and annotated bacterial genomes. Plasmid-encoded genes, previously isolated from low virulence strains of F. tularensis, were not identified. A total of 1289 potential coding ORFs were identified in the data set., An analysis of this data revealed 413 ORFs which would encode proteins with no homology to known proteins. ORFs which could encode proteins involved in amino acid and purine biosynthesis were also identified. These biosynthetic pathways provide targets for the construction of a defined attenuated mutant of F. tularensis for use as a vaccine against tularemia