Francisella tularensis is a gram-negative, facultative, intracellular bacterium that survives in mammals, arthropods, and amoebae; however, macrophages are considered the key cells in pathogenesis of tularemia in mammals. Understanding intracellular trafficking of F. tularensis within various host cells is indispensable to our understanding of bacterial ecology, intracellular adaptation to various hosts' microenvironments, and subversion of host cell defenses. Within mammalian and arthropod-derived cells, F. tularensis transiently resides within an acidic vacuole prior to escaping to the cytosol, where the bacteria replicate. In contrast, F. tularensis resides and replicates within non-acidified, membrane-bound vacuoles within the trophozoites of amoebae. The Francisella pathogenicity island (FPI) genes encode a type VI Secretion System (T6SS), which is indispensable for phagosomal escape of F. tularensis within mammalian and arthropod cells and for intravacuolar growth within amoeba. In this review, we discuss the divergent F. tularensis intracellular lifestyle in different hosts and its role in pathogenic evolution and intracellular proliferation within diverse hosts.

NKG2D is a potent activating receptor that is expressed on cytotoxic immune cells such as CD8 T and NK cells, where it promotes cytotoxicity after binding stress ligands on infected or transformed cells. On NK cell precursors NKG2D modulates proliferation and maturation. Previously, we observed that NKG2D deficiency affects peripheral B cell numbers. In this study, we show that NKG2D regulates B1a cell development and function. We find that mice deficient for NKG2D have a strong reduction of B1a cell numbers. As a result, NKG2D-deficient mice produce significantly less Ag-specific IgM Abs upon immunization with T cell–independent Ags, and they are more susceptible to Gram-negative sepsis. Klrk1^−/− B1a cells are also functionally impaired and they fail to provide protection against Francisella novicida upon adoptive transfer. Using mixed bone marrow chimeric mice, we show that the impact of NKG2D deficiency on B1a cell development is cell intrinsic. No changes in homeostatic turnover and homing of B cells were detectable, limiting the effects of NKG2D to modulation of the hematopoietic development of B1a cells. Using conditional ablation, we demonstrate that the effect of NKG2D on B1a cell development occurs at a developmental stage that precedes the common lymphoid progenitor. Our findings reveal an unexpected new role for NKG2D in the regulation of B1a cell development. The protective effects of this activating receptor therefore reach beyond that of cytotoxic cells, stimulating the immune system to fight bacterial infections by promoting development of innate-like B cells.

Francisella tularensis is a facultative intracellular bacterium that causes tularemia in humans and animals. Epidemiology of tularemia worldwide is often associated with water-borne transmission, which includes mosquitoes and amoebae as the potential host reservoirs of the bacteria in water environment. In vitro studies showed intracellular replication of F. tularensis within Acanthamoeba castellanii and Hartmanella vermiformis cells. While infection of amoeba by Legionella pneumophila has been shown to enhance infectivity of L. pneumophila the role of F. tularensis-infected protozoa in the pathogenesis of tularemia is not known. We used 6 h coculture of A. castellanii and F. novicida for investigation of the effect of inhaled amoeba on the pathogenesis of tularemia on in vivo model. Balb/c mice were infected intratracheally with F. novicida or with F. novicida-infected A. castellanii. Surprisingly, infection with F. novicida-infected A. castellanii did not lead to bronchopneumonia in Balb/c mice, and Francisella did not disseminate into the liver and spleen. Upon inhalation, F. novicida infects a variety of host cells, though neutrophils are the predominant cells early during infection in the lung infiltrates of pulmonary tularemia. The numbers of neutrophils in the lungs of Balb/c mice were significantly lower in the infection of mice with F. novicida-infected A. castellanii in comparison to group of mice infected only with F. novicida. These results demonstrate that following inoculation of mice with F. novicida-infected A. castellanii, mice did not develop tularemia.

The amoeba Dictyostelium discoideum has been used as a model organism to study host‐pathogen interaction in many intracellular bacteria. Francisella tularensis is a Gram‐negative, highly infectious bacterium that causes the zoonotic disease tularemia. The bacterium is able to replicate in different phagocytic and nonphagocytic cells including mammalian, amoebae, and arthropod cells. The aim of this study was to determine the optimal temperature and infection dose in the interaction of Francisella novicida with D. discoideum in order to establish a model of Francisella infection in the social amoeba. The amoeba cells were infected with a different multiplicity of infection (5, 10, and 100) and incubated at different temperatures (22, 25, 27, 30, and 37°C). The number of intracellular bacteria within D. discoideum, as well as cytotoxicity, was determined at 2, 4, 24, 48, and 72 hours after infection. Our results showed that the optimal temperature for Francisella intracellular replication within amoeba is 30°C with the MOI of 10. We can conclude that this MOI and temperature induced the optimal growth of bacteria in Dictyostelium with low cytotoxicity.