Francisella tularensis is a highly infectious, facultative intracellular bacterium which causes epidemics of tularemia in both humans and mammals at regular intervals. The natural reservoir of the bacterium is largely unknown, although it has been speculated that protozoa may harbor it. To test this hypothesis, Acanthamoeba castellanii was cocultured with a strain of F. tularensis engineered to produce green fluorescent protein (GFP) in a nutrient-rich medium. GFP fluorescence within A. castellanii was then monitored by flow cytometry and fluorescence microscopy. In addition, extracellular bacteria were distinguished from intracellular bacteria by targeting with monoclonal antibodies. Electron microscopy was used to determine the intracellular location of F. tularensis in A. castellanii, and viable counts were obtained for both extracellular and intracellular bacteria. The results showed that many F. tularensis cells were located intracellularly in A. castellanii cells. The bacteria multiplied within intracellular vacuoles and eventually killed many of the host cells. F. tularensis was found in intact trophozoites, excreted vesicles, and cysts. Furthermore, F. tularensis grew faster in cocultures with A. castellanii than it did when grown alone in the same medium. This increase in growth was accompanied by a decrease in the number of A. castellanii cells. The interaction between F. tularensis and amoebae demonstrated in this study indicates that ubiquitous protozoa might be an important environmental reservoir for F. tularensis.

A special form of a CuZn-superoxide dismutase with a high isoelectric point (hipI-SOD; EC 1.15.1.1) and hydrogen peroxide (H2O2) production were studied during the secondary cell wall formation of the inducible tracheary element cell-culture system of Zinnia elegans L. Confocal microscopy after labelling with 2',7'-dichlorofluorescin diacetate showed H2O2 to be located largely in the secondary cell walls in developing tracheary elements. Fluorescence-activated cell sorting analysis showed there were lower levels of H2O2 in the population containing tracheary elements when H2O2 scavengers such as ascorbate, catalase, and reduced glutathione were applied to the cell culture. Inhibitors of NADPH oxidase and SOD also reduced the amount of H2O2 in the tracheary elements. Furthermore, addition of these compounds to cell cultures at the time of tracheary element initiation reduced the amount of lignin and the development of the secondary cell walls. Analysis of UV excitation under a confocal laser scanning microscope confirmed these results. The expression of hipI-SOD increased as the number of tracheary elements in the cell culture increased and developed. Additionally, immunolocalization of a hipI-SOD isoform during the tracheary element differentiation showed a developmental build-up of the protein in the Golgi apparatus and the secondary cell wall. These findings suggest a novel hipI-SOD could be involved in the regulation of H2O2 required for the development of the secondary cell walls of tracheary elements.

Francisella tularensis is a small Gram-negative bacterium that causes tularemia in animals and man. The disease can be transmitted by handling of infected animals, by contaminated dust, by insect vectors, or by drinking contaminated water. In the present study cells of F. tularensis were subjected to extended storage in cold water devoid of carbon sources. Total cell counts remained constant throughout a 70-day period and beyond, while plate counts decreased to an undetectable level after 70 days. Attempts to resuscitate the cells were unsuccessful. Quantitative PCR targeting the 16S rDNA of F. tularensis showed an increase in variability after 25 days and the signal was lost after 45 days. Metabolic activity, measured by accumulation of rhodamine 123, declined to approximately 35% after a 140-day period. Analyses of substrate responsiveness of cells stored for 140 days in cold water showed that approximately 30% of the population increased in size after incubation in rich medium in the presence of nalidixic acid. Approximately 10(5) of these cells were injected intraperitoneally into mice. No signs or symptoms of tularemia were observed during 3 weeks. In addition, there was no evidence of stimulation of lymphocytes with F. tularensis as recall antigen. In conclusion, viable but non-culturable cells of F. tularensis are avirulent in mice, giving new insight into the ecological niche of this bacterium.

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Lipopolysaccharide (LPS) from the live vaccine strain of Francisella tularensis (F. tularensis LVS) was isolated and purified. The LPS did not stimulate lymphocytes from previously tularaemia‐vaccinated individuals or lymphocytes from nonprimed individuals. However, serum antibodies from tularaemia vaccines reacted with the LPS whereas virtually no reactivity was found with antibodies from individuals not exposed to F. tularensis LVS. Antibodies of immunoglobulin class M displayed the antibody reactivity predominantly. The LPS failed to induce the mononuclear cell‐derived cytokine interleukin‐1 and only low levels of tumour necrosis factor were detected. Furthermore, no LPS endotoxin properties were found in galactosamine‐treated mice or in the Limulus amoebocyte lysate assay. From these results it can be concluded that F. tularensis LVS possesses a lipopolysaccharide‐like molecule, which does not exhibit properties of a classical endotoxin.