Abstract
Fransicella tularensis (F.t.) is a facultative intracellular pathogen that escapes from the phagosome into the host cell cytosol in order to survive and proliferate. F.t. subspecies tularensis and holartica are the causative agents of tularemia infections in humans. It is a highly infectious disease with a dose as low as 10 colony forming units being able to cause severe disease upon infection. F.t. subspecies novicida (F.t.n.), which is not pathogenic to healthy humans, has been increasingly studied and used as a model organism for F.t. infections. This thesis aimed to establish a model for studying the F.t.n. intracellular cycle in murine bone marrow derived macrophages (BMM) and RAW 264.7 cells by transmission electron microscopy. Stereological analysis of transmission electron micrographs was used to investigate the kinetics of the intracellular life cycle of F.t.n., the effects of IFNγ-activation of macrophages, and the inhibition of lysosomal proteases on the ability of F.t.n. to escape from the phagosome. The interaction between the F.t.-containing vacuole and the endocytic compartment of the host cells was analyzed by immunogold labeling on Tokuyasu sections and by loading host cells with BSA-5 nm colloidal gold particles before infection. Optical tweezers were used to quantify bacterial adherence to a macrophage cell. In addition, the ultrastructural preservation of bacteria was compared using either chemical fixation by aldehydes and a standard resin embedding protocol or high-pressure freezing (HPF) and freeze substitution (FS). Our study showed that the bacterial load in RAW 264.7 was several folds higher compared to BMM, which argues that RAW 264.7 cells support the intracellular proliferation of F.t.n. better than BMM, which were very susceptible to the induction of cell death (pyroptosis) when bacteria escaped from the phagosome into the cytosol of the host cell. BSA-5 nm gold particles, LAMP-1 and cathepsin C, which represent markers of late endosomes/lysosomes, were localized to the majority of F.t.n. containing vacuoles 1 h and 4 h after the infection, which indicates that F.t.n. has a limited ability to modify phagosome maturation. IFNγ activated RAW 264.7 cells showed increased rate of cell death after the escape of bacteria into the cytosol, presumably due to increased susceptibility for the induction of pyroptosis. Inhibition of lysosomal proteases increased the fraction of cytosolic bacteria, which suggests that lysosomal cathepsins could degrade bacterial protein factors that mediate the phagosomal escape. Using optical tweezers we could demonstrate that Rhodococcus equi (R.e.) are two-fold more likely to adhere to a macrophage cell compared to F.t.n. We believe that with further optimization, optical tweezers could be a sensitive method to study interactions between bacteria and host cells at a single-cell level. Comparison between chemical fixation and HPF showed that F.t.n. is truly pleomorphic, that electron translucent halos around chemically fixed bacteria represents a shrinkage artefact, and that with chemical fixation the outer membrane is poorly immobilized and can be artefactually displaced in Tokuyasu sections. Collectively, our results contribute to the understanding of several aspects of the interaction between F.t. and its macrophage host cell, especially the uptake of bacteria, the phagosome maturation arrest and the phagosomal escape.