Abstract
Francisella tularensis is a Select Agent that causes the severe disease tularemia in humans and many animal species. The bacterium demonstrates rapid intracellular replication, however, macrophages can control its replication if primed and activation with IFN-γ is known to be essential, although alone not sufficient, to mediate such control. To further investigate the mechanisms that control intracellular F. tularensis replication, an in vitro co-culture system was utilized containing splenocytes obtained from naïve or immunized C57BL/6 mice as effectors and infected bone marrow-derived wild-type or chromosome-3-deficient guanylate-binding protein (GBP)-deficient macrophages. Cells were infected either with the F. tularensis live vaccine strain (LVS), the highly virulent SCHU S4 strain, or the surrogate for F. tularensis, F. novicida. Regardless of strain, significant control of the bacterial replication was observed in co-cultures with wild-type macrophages and immune splenocytes, but not in cultures with immune splenocytes and GBP chr3-deficient macrophages. Supernatants demonstrated very distinct, infectious agent-dependent patterns of 23 cytokines, whereas the cytokine patterns were only marginally affected by the presence or absence of GBPs. Levels of a majority of cytokines were inversely correlated to the degree of control of the SCHU S4 and LVS infections, but this was not the case for the F. novicida infection. Collectively, the co-culture assay based on immune mouse-derived splenocytes identified a dominant role of GBPs for the control of intracellular replication of various F. tularensis strains, regardless of their virulence, whereas the cytokine patterns markedly were dependent on the infectious agents, but less so on GBPs.
Highlights
Francisella tularensis is a facultative intracellular bacterium and the etiological agent of tularemia, a highly infectious disease affecting humans and a wide range of animals
Co-cultures were established by overlaying bone marrow-derived macrophages (BMDM) monolayers infected with either the F. novicida, live vaccine strain (LVS), or SCHU S4 strains with splenocytes from naïve or DclpB-immune mice and bacterial replication was followed for a period of 72 h
In cultures with wild-type BMDM, the outcome of the F. novicida and LVS infections was rather similar and addition of immune splenocytes resulted in approximately 1.7 log10 CFU lower numbers than in cultures with naïve splenocytes (P < 0.01 for F. novicida and P < 0.001 for LVS; Figures 1A, B)
Summary
Francisella tularensis is a facultative intracellular bacterium and the etiological agent of tularemia, a highly infectious disease affecting humans and a wide range of animals. F. tularensis is highly contagious, since its infectious dose is very low and it may be spread via aerosol. These characteristics, together with high virulence, have led to its classification as a Tier 1 Select Agent, along with other potential agents of bioterrorism. Two subspecies cause human disease, subspecies tularensis, with high mortality resulting if untreated, and subspecies holarctica, which despite lower virulence still can cause serious illness in humans. The closely related species F. novicida is an often used surrogate for F. tularensis and a very rare human pathogen, it is virulent for many animal species and highly virulent in the frequently used mouse model of tularemia
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