01, and *** P < 0.0001). Panel C: IL-1β and IL-1α concentrations in BALF collected from mice 48 hrs following intranasal Duvelisib nmr infection with either WT or galU mutant strains of FT
were determined via multiplex cytokine analysis. Statistical analyses were performed via unpaired t tests and two-tailed p values are indicated. Cytotoxicity of the galU mutant In light of the findings that mutation of the galU gene resulted in altered kinetics of innate signaling and earlier production of IL-1β than was observed with WT FT, we speculated that the galU mutant might induce death of the host cell more CH5183284 solubility dmso rapidly than WT FT. To investigate this possibility, we evaluated the relative abilities of the galU mutant and WT FT strains to kill their host cells in vitro. A macrophage-like cell line (J774) was infected with either the galU mutant or WT FT strains at an MOI of 100 and incubated for 24 hours. LDH activity in the culture medium was then determined as a measure of host cell death. A significantly higher amount of LDH activity was measured in the supernatants of J774 cells that had been infected
Proteasome purification with the galU mutant compared to those infected with WT FT (p < 0.0001), indicating that the galU mutant was hyper-cytotoxic. Complementation of the galU mutation in trans partially restored the cytotoxicity phenotype. For comparative purposes, a wbtA mutant strain of FT was also included and was shown to have cytotoxicity characteristics similar to those of WT FT (Figure 7). Figure 7 Mutation of the galU gene increases cytotoxicity of FT. Murine macrophage-like cells (J774) were infected with the WT, galU mutant, the galU-complemented, or wbtA mutant (O-antigen-deficient) FT LVS strains at an MOI of 100. Host cell death was determined by measuring LDH released from infected cells 24-hours post-infection. crotamiton All data points
represent the mean (± SEM) of triplicate samples and the data shown is representative of three experiments of similar design. Statistical analyses were performed via one-way ANOVA with a Bonferroni multiple comparisons post-test (*** indicates a p-value of <0.0001). Immunization with the galU mutant confers immunity to WT FT challenge Because infection with the galU mutant elicited a robust innate immune response and infected mice were able to clear the infection, we assessed the efficacy of the galU mutant strain as a live attenuated vaccine strain. Two months following the initial inoculation, mice that survived infection with the galU mutant, as well as a naïve group of mice, were challenged with a large dose of WT (50 × LD50) via the intranasal route and were monitored for survival. The galU mutant-immunized mice experienced transient weight loss following challenge, but displayed no other visible symptoms of tularemic disease and survived the infection. In contrast, each of the naive mice displayed the typical visible signs of tularemia (lack of grooming, hunched posture, reduced motor activity, etc.