From this analysis, all of the ∆yscN Osimertinib colonies examined (n = 50) still maintained pLcr. The PCR controls for this experiment included colonies of the parental strain CO92 (n = 10) which maintained the plasmid, and colonies of the pLcr− strain (n = 10) which served as a negative control and did not amplify a PCR product (data not shown). The existence of T3SS in various bacterial species, each reliant on only a single ATPase for virulence factor delivery, suggests a critical role for T3SS ATPases. The introduction

of a deletion within the gene encoding for the Y. pestis CO92 YscN ATPase is expected to at least decrease virulence factor secretion and possibly attenuate virulence. Indeed, the deletion within the yscN gene led to attenuation following s.c. mice challenges. In the initial virulence testing, groups of mice (n = 3) were challenged s.c. with either 4.44 × 104

or 4.44 × 106 CFU of the ΔyscN mutant. However, after following the mice for 21 days, none succumbed to infection (data not shown). In contrast, based upon previous data, the s.c. LD50 value for the wild-type CO92 strain is 1.9 CFU (Welkos et al., 1995) and time to death with 40 CFUs is approximately 5.9 days (Bozue et al., 2011). The yscN deletion AZD4547 in vivo was performed in-frame, and the RT-PCR data demonstrated that a polar effect on downstream genes did not occur. However, to confirm that attenuation was due specifically to the mutation of the yscN gene, the mutant was complemented in trans with a functional yscN gene on pWKS30 to form pYSCN. Mice were challenged s.c. at two different doses, as indicated in Fig. 3, with the wild-type CO92 parental strain, ΔyscN mutant, ΔyscN + pWKS30, or the complemented mutant (ΔyscN + pYSCN). As expected, no differences in survival were noted between the Fluorometholone Acetate wild-type and complemented strain at either dose (Fig. 3). In contrast,

no mice succumbed to infection when challenged with ΔyscN or ΔyscN + pWKS30 (Fig. 3). Therefore, loss of virulence was due specifically to the deletion of the yscN gene. In addition, no CFUs were recovered from the spleens of three mice from both the high and low ΔyscN challenged groups collected 21 days postchallenge (data not shown). The attenuation of the Y. pestis ΔyscN strain suggested the possible use of the strain as a live vaccine. In the current work, we asked whether inoculation with the ΔyscN strain of varying doses would be sufficient to provide protection against the fully virulent Y. pestis CO92 strain. The mice were immunized s.c. twice with doses of the mutant strain ranging from 102 to 107 CFU or KPBS alone. The mice survived the immunization regimen of all doses of the ΔyscN strain (Table 1). On the 60th day following the initial immunization, the mice were challenged s.c. with 180 CFU of the wild-type CO92 strain and their survival was monitored (Fig. 4 and Table 1).