The type IV pili component PilO is a virulence determinant of Francisella novicida.

Francisella tularensis is a highly pathogenic intracellular bacterium that causes the disease tularemia. While its ability to replicate within cells has been studied in much detail, the bacterium also encodes a less characterised type 4 pili (T4P) system. T4Ps are dynamic adhesive organelles identified as major virulence determinants in many human pathogens. In F. tularensis, the T4P is required for adherence to the host cell, as well as for protein secretion. Several components, including pilins, a pili peptidase, a secretin pore and two ATPases, are required to assemble a functional T4P, and these are encoded within distinct clusters on the Francisella chromosome. While some of these components have been functionally characterised, the role of PilO, if any, still is unknown. Here, we examined the role of PilO in the pathogenesis of F. novicida. Our results show that the PilO is essential for pilus assembly on the bacterial surface. In addition, PilO is important for adherence of F. novicida to human monocyte-derived macrophages, secretion of effector proteins and intracellular replication. Importantly, the pilO mutant is attenuated for virulence in BALB/c mice regardless of the route of infection. Following intratracheal and intradermal infection, the mutant caused no histopathology changes, and demonstrated impaired phagosomal escape and replication within lung liver as well as spleen. Thus, PilO is an essential virulence determinant of F. novicida.

Atg5-Deficient Mice Infected with Francisella tularensis LVS Demonstrate Increased Survival and Less Severe Pathology in Internal Organs.

Francisella tularensis is a highly virulent intracellular pathogen that proliferates within various cell types and can infect a multitude of animal species. Francisella escapes the phagosome rapidly after infection and reaches the host cell cytosol where bacteria undergo extensive replication. Once cytosolic, Francisella becomes a target of an autophagy-mediated process. The mechanisms by which autophagy plays a role in replication of this cytosolic pathogen have not been fully elucidated. In vitro, F. tularensis avoids degradation via autophagy and the autophagy process provides nutrients that support its intracellular replication, but the role of autophagy in vivo is unknown. Here, we investigated the role of autophagy in the pathogenesis of tularemia by using transgenic mice deficient in Atg5 in the myeloid lineage. The infection of Atg5-deficient mice with Francisella tularensis subsp. holarctica live vaccine strain (LVS) resulted in increased survival, significantly reduced bacterial burden in the mouse organs, and less severe histopathological changes in the spleen, liver and lung tissues. The data highlight the contribution of Atg5 in the pathogenesis of tularemia in vivo.

Increased Sensitivity of Amoeba-Grown Francisella Species to Disinfectants.

Francisella tularensis is a highly infectious, intracellular bacterium and it is the causative agent of tularemia. The bacterium has been isolated from more than 250 species, including protozoa. Previous studies have shown that the growth of Legionella pneumophila within the amoeba results in a dramatic increase in the resistance to disinfectants. Since Francisella persists in the environment for years, this study investigates whether Acanthamoeba castellanii-grown F. novicida exhibits an alteration in the resistance to disinfectants. The disinfectants used are didecyldimethylammonium chloride (DDAC) combined with isopropyl alcohol (D1), benzalkonium chloride combined with DDAC and formic acid (D2), and polyhexamethylene biguanide (PHMB, D3). The effect of disinfectants on the bacterial viability is determined by a colony-forming unit (CFU), by transmission electron microscopy (TEM), by fluorescence microscopy, and the damage of the bacterial membrane. Our data has shown that only a one-log10 loss in bacterial viability is exhibited upon treatment of agar-grown Francisella, while in amoeba-grown Francisella there was a three-log10 difference with D3. The D1 disinfectant sterilized the bacteria within 10 s. The treatment of agar-grown F. novicida with D2 reduces bacterial viability by seven-log10 within 10 s and 15 min, respectively. Surprisingly, the treatment of amoeba-grown F. novicida with D2 results in a total loss of bacterial viability. In conclusion, A. castellanii-grown F. novicida is more susceptible to many disinfectants.

Epidemiologic and Epizootic Data of Tularemia in the Past and in the Recent History in Croatia.

Tularemia is a zoonotic disease caused by Francisella tularensis. A large number of recent studies have provided an update on the disease characteristics and the distribution across Europe. In Croatia, most of the clinical cases, as well as the reports of the disease in animals, date from the 20th century. In that period, epidemic and epizootic research had given detailed information about endemic regions and their characteristics, including suspected animal hosts and vectors. The region along the middle course of the Sava River, called Middle Posavina, is described as an endemic region, i.e., a "natural focus" of tularemia, in Croatia. In the 21st century, cases of human tularemia are being reported sporadically, with ulceloglandular, oropharyngeal and typhoid forms of disease. A majority of the described cases are linked with the consumption of contaminated food or water. The disease outbreaks still occur in areas along the course of the river Sava and in northwest Croatia. In this review article, we have summarized epidemiologic and epizootic data of tularemia in the past and in recent Croatian history.

Optimisation of External Factors for the Growth of Francisella novicida within Dictyostelium discoideum.

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.

The role of B cells in an early immune response to Mycobacterium bovis.

Mycobacterium tuberculosis is the main etiological agent of tuberculosis. The Bacillus Calmette-Guérin (BCG) microbes that are primarily used as a vaccine against tuberculosis also constitute the dominant infection model for studying the interaction of mycobacteria with the host cell types. The majority of interaction experiments have been conducted using macrophages and monocytes as prototype phagocyte cell types. Here, we report that M. bovis BCG infects mouse primary B cells as well as human B cell line. The complement receptors, along with B cell receptors, are engaged in the process of bacterial entry into the host B cells. Once inside the B cells, the intracellular trafficking of BCG follows the complete endocytic pathway of the ingested particles, which is in contrast to the events taking place during ingestion of BCG by macrophages. In vivo infection of mice with M. bovis BCG activated peritoneal as well as splenic B cells to produce proinflammatory cytokines. This paper further supports the evidence that B cells are involved in a host's early interactions with intracellular bacterial pathogens and participate in the induction of innate defense responses.

Isolation of F. novicida-Containing Phagosome from Infected Human Monocyte Derived Macrophages.

Francisella is a gram-negative bacterial pathogen, which causes tularemia in humans and animals. A crucial step of Francisella infection is its invasion of macrophage cells. Biogenesis of the Francisella-containing phagosome (FCP) is arrested for ~15 min at the endosomal stage, followed by gradual bacterial escape into the cytosol, where the microbe proliferates. The crucial step in pathogenesis of tularemia is short and transient presence of the bacterium within phagosome. Isolation of FCPs for further studies has been challenging due to the short period of time of bacterial residence in it and the characteristics of the FCP. Here, we will for the first time present the method for isolation of the FCPs from infected human monocytes-derived macrophages (hMDMs). For elimination of lysosomal compartment these organelles were pre-loaded with dextran coated colloidal iron particles prior infection and eliminated by magnetic separation of the post-nuclear supernatant (PNS). We encountered the challenge that mitochondria has similar density to the FCP. To separate the FCP in the PNS from mitochondria, we utilized iodophenylnitrophenyltetrazolium, which is converted by the mitochondrial succinate dehydrogenase into formazan, leading to increased density of the mitochondria and allowing separation by the discontinuous sucrose density gradient ultracentrifugation. The purity of the FCP preparation and its acquisition of early endosomal markers was confirmed by Western blots, confocal and transmission electron microscopy. Our strategy to isolate highly pure FCPs from macrophages should facilitate studies on the FCP and its biogenesis.

NKG2D Promotes B1a Cell Development and Protection against Bacterial Infection.

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.

Phenotypic characterization of the Francisella tularensis ΔpdpC and ΔiglG mutants.

Several bacterial pathogens interact with their host through protein secretion effectuated by a type VI secretion system (T6SS). Francisella tularensis is a highly pathogenic intracellular bacterium that causes the disease tularemia. Proteins encoded by the Francisella pathogenicity island (FPI), which constitute a type VI secretion system, are essential for the virulence of the bacterium and a key mechanism behind this is the escape from the phagosome followed by productive cytosolic replication. It has been shown that T6SS in Francisella is distinct since all putative substrates of F. tularensis T6SS, except for VgrG, are unique to the species. Many of the FPI proteins are secreted into the macrophage cytosol and this is dependent on the functional components of DotU, VgrG, IglC and IglG. In addition, PdpC seems to have a regulatory role for the expression of iglABCD. Since previous results showed peculiar phenotypes of the ΔpdpC and ΔiglG mutants in mouse macrophages, their unique behavior was characterized in human monocyte-derived macrophages (HMDM) in this study. Our results show that both ΔpdpC and ΔiglG mutants of the live vaccine strain (LVS) of F. tularensis did not replicate within HMDMs. The ΔpdpC mutant did not escape from the Francisella containing phagosome (FCP), neither caused cytopathogenicity in primary macrophages and was attenuated in a mouse model. Interestingly, the ΔiglG mutant escaped from the HMDMs FCP and also caused pathological changes in the spleen and liver tissues of intradermally infected C57BL/6 mice. The ΔiglG mutant, with its unique phenotype, is a potential vaccine candidate.

F. novicida-Infected A. castellanii Does Not Enhance Bacterial Virulence in Mice.

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.