Guanylate-Binding Proteins Are Critical for Effective Control of Francisella tularensis Strains in a Mouse Co-Culture System of Adaptive Immunity

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 GBPchr3-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.

Comparative study of GBP recruitment on two cytosol-dwelling pathogens, Francisella novicida and Shigella flexneri highlights differences in GBP repertoire and in GBP1 motif requirements

“…Consequently, S. flexneri escapes from GBPs-mediated growth restriction (14,15,21). Francisella tularensis, the agent of tularemia, is another professional cytosolic Gramnegative pathogen that escapes GBP-mediated growth restriction although to different extents depending on the subspecies considered (16,29). F. tularensis can infect a large number of host cells but has a particular tropism for phagocytic cells, including macrophages (30).…”

Bacterial factors drive the differential targeting of Guanylate Binding Proteins toFrancisellaandShigella

“…Consequently, S. flexneri escapes from GBPs-mediated growth restriction (14, 15, 21). Francisella tularensis, the agent of tularemia, is another professional cytosolic Gram-negative pathogen that escapes GBP-mediated growth restriction although to different extents depending on the subspecies considered (16, 29). F. tularensis can infect a large number of host cells but has a particular tropism for phagocytic cells, including macrophages (30).…”

Analyses of human immune responses to Francisella tularensis identify correlates of protection

“…This was first demonstrated for the closely related bacterium F. novicida and subsequently also for the LVS strain using infection of mononuclear cells; however, control of the highly virulent SCHU S4 strain was not observed in the model ( 48 ). Later, it was demonstrated that control of infection with each of the three F. tularensis strains occurred in a mouse co-culture model, but, again, control was critically dependent on GBPs ( 49 ). Thus, the evidence from animal models indicate that the control of highly virulent strains is distinct from that of attenuated F. tularensis strains and demonstrates that the use of such strains in the models is necessary to identify relevant correlates of immunity and protection.…”