Abstract
Francisella tularensis is a highly virulent intracellular bacterium and cell-mediated immunity is critical for protection, but mechanisms of protection against highly virulent variants, such as the prototypic strain F. tularensis strain SCHU S4, are poorly understood. To this end, we established a co-culture system, based on splenocytes from naïve, or immunized mice and in vitro infected bone marrow-derived macrophages that allowed assessment of mechanisms controlling infection with F. tularensis. We utilized the system to understand why the clpB gene deletion mutant, ΔclpB, of SCHU S4 shows superior efficacy as a vaccine in the mouse model as compared to the existing human vaccine, the live vaccine strain (LVS). Compared to naïve splenocytes, ΔclpB-, or LVS-immune splenocytes conferred very significant control of a SCHU S4 infection and the ΔclpB-immune splenocytes were superior to the LVS-immune splenocytes. Cultures with the ΔclpB-immune splenocytes also contained higher levels of IFN-γ, IL-17, and GM-CSF and nitric oxide, and T cells expressing combinations of IFN-γ, TNF-α, and IL-17, than did cultures with LVS-immune splenocytes. There was strong inverse correlation between bacterial replication and levels of nitrite, an end product of nitric oxide, and essentially no control was observed when BMDM from iNOS−/− mice were infected. Collectively, the co-culture model identified a critical role of nitric oxide for protection against a highly virulent strain of F. tularensis.
Highlights
Francisella tularensis is a highly virulent facultative intracellular bacterium causing the severe disease tularemia in many mammalian species (Sjöstedt, 2007)
Splenocytes from mice immunized with live vaccine strain (LVS) or clpB were stimulated with formalin-killed F. tularensis antigen in vitro to measure the recall response of the T-cells with regard to proliferation and expression of cytokines
Both LVS- and clpB-immune Balb/c splenocytes responded with very prominent proliferative responses to the antigen and the responses were stronger, not significantly stronger, of clpB- compared to LVS-immune splenocytes (P = 0.16; Figure 1A)
Summary
Francisella tularensis is a highly virulent facultative intracellular bacterium causing the severe disease tularemia in many mammalian species (Sjöstedt, 2007). Two subspecies are common human pathogens, subspecies tularensis (type A), which causes disease with high mortality if untreated, and the less aggressive subspecies holarctica (type B), which despite its lower virulence. There is a need for development of more efficacious Francisella vaccines and previously, we analyzed if defined mutants of SCHU S4 (type A) could serve as such vaccine candidates and identified that the clpB mutant conferred superior efficacy compared to LVS, despite that the former was more attenuated (Kadzhaev et al, 2009; Conlan et al, 2010; Ryden et al, 2012) It encodes an AAA+ chaperone, the exact function in F. tularensis is still unknown
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