Contribution of citrulline ureidase to Francisella tularensis strain Schu S4 pathogenesis

The citrulline ureidase (CTU) activity has been shown to be associated with highly virulentFrancisella tularensisstrains, including Schu S4, while it is absent in avirulent or less virulent strains. A definitive role of thectugene in virulence and pathogenesis ofF. tularensisSchu 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 theF. tularensisSchu S4 genome. In the present study, we evaluated the contribution of thectugene in the virulence of category A agentF. tularensisSchu S4 by generating a nonpolar deletion mutant, the Δctumutant. The deletion of thectugene resulted in loss of CTU activity, which was restored by transcomplementing thectugene. The Δctumutant 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 Δctumutant was further tested for its virulence attributes in a mouse model of respiratory tularemia. Mice infected intranasally with the Δctumutant 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 Δctumutant was also associated with significantly lower histopathological scores in the lungs. Mice infected with the Δctumutant 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 thatctucontributes to intracellular survival, in vivo growth, and pathogenesis. However,ctuis not an absolute requirement for the virulence ofF. tularensisSchu S4 in mice.