Valentina Marečić

The Divergent Intracellular Lifestyle of Francisella tularensis in Evolutionarily Distinct Host Cells

Nonpathogenic bacteria are taken up by host cells into vacuoles or phagosomes that are processed through the endocytic pathway, through which the vacuoles mature and fuse to the lysosomes, in which the bacteria are degraded. To avoid this fate within phagocytic cells, intracellular pathogens have evolved different strategies to survive and evade phagosome–lysosome fusion [1]. Understanding the mechanisms by which pathogens manipulate vesicle trafficking in different hosts is extremely important for understanding the ability of various pathogens to cause disease and is essential for designing novel and effective strategies for prevention and therapeutic intervention. Francisella tularensis is a gram-negative, highly infectious, facultative intracellular bacterium that causes the zoonotic disease tularemia. The genus Francisella contains five species: F. tularensis, F. philomiragia, F. hispaniensis, F. noatunensis, and F. novicida [2,3]. Two subspecies of F. tularensis, tularensis (Type A) and holarctica (Type B), cause most cases of the illness in humans. F. novicida causes disease only in immunocompromised persons, but is highly virulent in mice [3]. However, it is important to note that in comparison to F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, F. novicida elicits a different immune response in the host [2]. Humans acquire infection by several routes, including direct contact with infected animals, ingestion of water or food contaminated by infected animals, exposure to infected arthropod vectors, or by inhalation of infective aerosols, resulting in pneumonic, oropharyngeal, glandular, ulceroglandular, or oculoglandular tularemia [4]. Considering the ease of dissemination and high infectivity, F. tularensis subsp. tularensis and F. tularensis subsp. holarctica have been classified by the Centers for Disease Control and Prevention (CDC) as Tier 1 select agents. The existence of Francisella in the environment is divided into two cycles: the terrestrial cycle and aquatic cycle [5]. Small rodents, hares, and arthropods play a major role in the terrestrial cycle, while rodents associated with water are important in the water cycle [4,5]. Organisms such as ticks, flies, and mosquitoes are considered vectors of tularemia transmission to mammals [6]. Although it causes disease in various animal species, no animal has been identified as a main reservoir of this pathogen. F. tularensis subsp. holarctica and F. novicida have a strong association with freshwater environments, free-living amoeba, and biofilms [7,8]. Since mosquito larvae can feed on aquatic protozoa, they may be infected with F. tularensis during development in their natural aquatic environment [7]. The effect of wars, natural disasters, climate change, and global warming will probably have an impact on increased incidences of tularemia [9].

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.

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.

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.