Viable bacteria were then enumerated by dilution plating. 100% viability was defined as the number of bacteria recovered from PBS containing no serum (0% FFP), and results were plotted as the mean (± SEM) of triplicate samples. Statistical analyses were performed via one-way ANOVA and statistically significant differences (P < 0.0001) are indicated (***). To further investigate whether the galU gene resulted in gross change(s) to the outer envelope of FT, experiments were performed to measure the relative sensitivity of galU mutant and WT FT to serum components. The Semaxanib in vivo galU mutant, WT, and galU-complemented
strains of FT all displayed a similar pattern of serum sensitivity. In contrast, an O-antigen-deficient (ΔwbtA mutant) strain of FT was highly sensitive to serum. Interestingly, the galU, WT, and galU-complemented strains were equally sensitive to heat-inactivated serum, while Mizoribine the wbtA mutant strain displayed no sensitivity to serum that had been heat inactivated (Figure 5C). IL-1 expression/activation induced by the galU mutant vs. WT FT Activation of the AIM2 inflammasome and production of IL-1β and IL-18 are known to be a critical component of the innate immune response to FT infection . We compared the kinetics of IL-1β production following
infection (in vitro and in vivo) with either the galU mutant or WT strain of FT. RNase protection analysis Edoxaban revealed that IL-1β mRNA levels (as well as those of several other cytokines) were similar in bone marrow-derived dendritic cells (BMDC) that had been infected for 8 h with either the galU mutant, WT, or galU-complemented strains of FT (Figure 6A), confirming
the comparable abilities of the galU mutant and WT strains to stimulate TLR-mediated events such as cytokine expression. However, 24 h after infection of a macrophage-like cell line (THP-1) or BMDCs with the galU mutant, the amount of IL-1β released into culture supernatants was significantly higher (p < 0.0001 and p < 0.01, respectively) than was observed selleck chemicals llc following infection with WT FT (Figure 6B). The galU mutant also induced accelerated kinetics of IL-1β protein production in vivo (Figure 6C). Moreover, the kinetics of IL-1α protein production is more rapid following infection with the galU mutant strain of FT (Figure 6C). Figure 6 galU mutant and WT FT differentially induce cleavage of pro-IL-1β to the active IL-1β form. Panel A: BMDC were infected with WT, galU mutant, and galU-complemented strains of FT at the indicated MOI, and total RNA was extracted 8 h later and subjected to RNase protection analysis.