Determining the role of guanylate-binding proteins for host defense against Francisella tularensis

Sammanfattning: Francisella tularensis is a highly virulent, intracellular bacterium and the causative agent of the human disease tularemia. This is a zoonotic, often vector-borne disease. Due to its intracellular nature, F. tularensis can infect many cell types, but of special relevance is its ability to infect monocytic cells and avoid their otherwise potent antimicrobial effects. Monocytic cells can; however, control infection after activation with IFN-γ, but the molecular mechanisms behind this control are not well understood. Recently, guanylate-binding proteins (GBPs) have been identified as crucial for the control of intracellular F. tularensis and many other bacteria, viruses, and parasites. They represent a vast family of interferon-inducible proteins, but it is incompletely understood how their ubiquitous abilities to control diverse types of infections are executed. The overall aim of the thesis was to obtain a better understanding of how GBPs execute the control of infection caused by Francisella and how the bacterium counteracts the bactericidal effects of the GBPs and of other immune mediators. To this end, the responses of bone marrow-derived murine macrophages (BMDM) to Francisella was one model investigated and the other employed a co-culture system whereby BMDM were infected and to the cultures immune cells from vaccinated mice were added. To comprehensively understand the host-pathogen interaction, a variety of Francisella strains were utilized; the highly virulent SCHU S4 strain, the human live vaccine strain (LVS), and the widely used surrogate for F. tularensis, the low virulent F. novicida. All strains have similar capability of intracellular multiplication in BMDM, however, activation of the microbicidal ability of BMDM with IFN-γ, significant control of infection was observed for the LVS and F. novicida strains, whereas there was no control of the SCHU S4 infection. The control of the former strains was GBP-dependent, despite that no differences in GBP transcription or translation were observed in the infected cell cultures. Patterns of 18 cytokines very clearly discriminated the different types of infections and high levels were generally observed in F. novicida-infected cultures and very low levels in SCHU S4-infected cultures. Co-infection with F. novicida and SCHU S4 led to significant control of both strains and in these cultures, a majority of cytokines showed intermediate or high levels. A critical component in the immune recognition of Francisella is AIM2, which is a core constituent of a special form of inflammasome, a cytoplasmic multimeric complex. We determined that AIM2-deficient BMDM, despite the central role of AIM2 for immune recognition of F. novicida and LVS, still controlled infection with either of the two strains after activation with IFN-γ. Again, no control of the virulent strain SCHU S4 was observed. The co-culture system revealed further complexity beyond that of the BMDM model. Utilizing splenocytes obtained from immunized C57BL/6 mice as effectors in cultures with BMDM infected with either of the three Francisella strains, we observed that regardless of strain, significant control of replication occurred with wild-type macrophages and immune splenocytes, even for the highly virulent SCHU S4 strain, but not in cultures with immune splenocytes and GBP-deficient macrophages. Supernatants from the cultures demonstrated very distinct patterns for each of the three infections. Thus, the co-culture assay identified, as for the BMDM model, a crucial role of GBPs for the control of intracellular replication of Francisella, however, in contrast to the BMDM model, the co-culture conferred significant control of SCHU S4 infection.Collectively, our studies demonstrate a very important role of GBPs for the IFN-γ-dependent control of Francisella infection, with the notable exception of the highly virulent strain SCHU S4. A GBP-mediated control of SCHU S4 was; however, observed in the co-culture system, thereby identifying additional bactericidal mechanisms, besides those that are IFN-γ-dependent. We also demonstrate that the inflammatory potential of Francisella strains is correlated to their virulence, most notable is the almost complete lack of inflammatory response during infection with the highly virulent SCHU S4 strain, but this anti-inflammatory capacity was counteracted by the strong pro-inflammatory property of F. novicida during co-infection.