Contribution of Citrulline Ureidase toFrancisella tularensisStrain Schu S4 Pathogenesis

Abstract

The citrulline ureidase (CTU) activity has been shown to be associated with highly virulent Francisella tularensis strains, including Schu S4, while it is absent in avirulent or less virulent strains. A definitive role of the ctu gene in virulence and pathogenesis of F. tularensis Schu S4 has not been assessed; thus, an understanding of the significance of this phenotype is long overdue. CTU is a carbon-nitrogen hydrolase encoded by the citrulline ureidase (ctu) gene (FTT0435) on the F. tularensis Schu S4 genome. In the present study, we evaluated the contribution of the ctu gene in the virulence of category A agent F. tularensis Schu S4 by generating a nonpolar deletion mutant, the ⌬ctu mutant. The deletion of the ctu gene resulted in loss of CTU activity, which was restored by transcomplementing the ctu gene. The ⌬ctu mutant did not exhibit any growth defect under acellular growth conditions; however, it was impaired for intramacrophage growth in resting as well as gamma interferon-stimulated macrophages. The ⌬ctu mutant was further tested for its virulence attributes in a mouse model of respiratory tularemia. Mice infected intranasally with the ⌬ctu mutant showed significantly reduced bacterial burden in the lungs, liver, and spleen compared to wild-type (WT) Schu S4-infected mice. The reduced bacterial burden in mice infected with the ⌬ctu mutant was also associated with significantly lower histopathological scores in the lungs. Mice infected with the ⌬ctu mutant succumbed to infection, but they survived longer and showed significantly extended median time to death compared to that shown by WT Schu S4-infected mice. To conclude, this study demonstrates that ctu contributes to intracellular survival, in vivo growth, and pathogenesis. However, ctu is not an absolute requirement for the virulence of F. tularensis Schu S4 in mice.

Identification of a Novel Francisella tularensis Factor Required for Intramacrophage Survival and Subversion of Innate Immune Response

“…For construction of the ⌬FTT0831c mutant, the entire coding region of the FTT0831c gene was deleted by employing an approach described earlier (25,26). For transcomplementation, the full-length FTT0831c gene was amplified by PCR and cloned in pKK214 vector downstream of GroEL promoter of F. tularensis following our recently published protocol (26,27).…”

“…Macrophage Invasion and Replication Assays-Gentamicin protection assays were performed as described earlier (26). In macrophage assays involving live or UV-killed WT F. tularensis LVS, SchuS4, FTL_0325, or ⌬FTT0831c mutant and its transcomplemented strain, a multiplicity of infection (m.o.i.)…”

Identification of Francisella tularensis Live Vaccine Strain CuZn Superoxide Dismutase as Critical for Resistance to Extracellularly Generated Reactive Oxygen Species

“…An allelic replacement method was used to construct an in-frame sodC gene deletion mutant (⌬sodC) and sodB ⌬sodC mutants of F. tularensis LVS (13). For construction of the ⌬sodC mutant, the entire 557-bp coding region of the sodC gene was deleted employing an approach described earlier (23). Briefly, the regions approximately 750 and 650 bp up-and downstream of the sodC gene were PCR amplified.…”

“…The sodB ⌬sodC double mutation was confirmed by multiplex PCR followed by digestion of the products with the PstI restriction enzyme. The mutant cultures were stored at Ϫ80 C. A pKK214::gfp vector expressing green fluorescent protein under the control of the groEL promoter of F. tularensis was used for transcomplementation according to a recently reported protocol (23). The 557-bp sodC gene along with its upstream 100-bp region, and the kanamycin resistance gene of the pKK214 vector were amplified in separate PCRs using primer pairs detailed in Table 1.…”

Identification of Mechanisms for Attenuation of the FSC043 Mutant of Francisella tularensis SCHU S4

“…tularensis strains. Several such vaccine candidates have been characterized more recently (11)(12)(13)(14)(15)(16)(17)(18).…”