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Francisella tularensis is a gram-negative facultative intracellular bacterium that resists harsh environments. Several outbreaks of tularemia are linked to the consumption and contact with spring water. The number of F. tularensis in some waters is high, while in others, this bacterium does not survive. Except organic compounds, metals could be important for the survival of F. tularensis in water. Some Francisella strains showed the association with amoeba, which may act as the environmental reservoir. This study was aimed at following the role of metal ions and/or amoeba in the existence and replication of F. novicida in spring waters by growth kinetics, acquisition of metals, and ultrastructural analyses of bacteria. The bacteria showed a longer survival in water with higher initial concentrations of Mn and Zn. Although Mn and Zn were necessary for the survival of F. novicida, the results also showed that the bacterium does not grow in water with high levels of Zn. In contrast, high levels of Mn did not have such a negative effect on the survival of this bacterium in water. In addition, while F. novicida benefits presence of amoeba in spring water, the number of amoebae is decreasing in a coculture model with F. novicida.

Intracellular proliferation of Legionella pneumophila within a vacuole in human alveolar macrophages is essential for manifestation of Legionnaires’ pneumonia. Intravacuolar growth of the pathogen is totally dependent on remodeling the L. pneumophila-containing vacuole (LCV) by the ER and on its evasion of the endosomal-lysosomal degradation pathway., Diversion of the Legionella pneumophila-containing vacuole (LCV) from the host endosomal-lysosomal degradation pathway is one of the main virulence features essential for manifestation of Legionnaires’ pneumonia. Many of the ∼350 Dot/Icm-injected effectors identified in L. pneumophila have been shown to interfere with various host pathways and processes, but no L. pneumophila effector has ever been identified to be indispensable for lysosomal evasion. While most single effector mutants of L. pneumophila do not exhibit a defective phenotype within macrophages, we show that the MavE effector is essential for intracellular growth of L. pneumophila in human monocyte-derived macrophages (hMDMs) and amoebae and for intrapulmonary proliferation in mice. The mavE null mutant fails to remodel the LCV with endoplasmic reticulum (ER)-derived vesicles and is trafficked to the lysosomes where it is degraded, similar to formalin-killed bacteria. During infection of hMDMs, the MavE effector localizes to the poles of the LCV membrane. The crystal structure of MavE, resolved to 1.8 Å, reveals a C-terminal transmembrane helix, three copies of tyrosine-based sorting motifs, and an NPxY eukaryotic motif, which binds phosphotyrosine-binding domains present on signaling and adaptor eukaryotic proteins. Two point mutations within the NPxY motif result in attenuation of L. pneumophila in both hMDMs and amoeba. The substitution defects of P78 and D64 are associated with failure of vacuoles harboring the mutant to be remodeled by the ER and results in fusion of the vacuole to the lysosomes leading to bacterial degradation. Therefore, the MavE effector of L. pneumophila is indispensable for phagosome biogenesis and lysosomal evasion.

Diversion of the Legionella pneumophila- containing vacuole (LCV) from the host endosomal- lysosomal degradation pathway is one of the main virulence features essential for manifestation of Legionnaires' pneumonia. Many of the ∼350 Dot/Icm-injected effectors identified in L. pneumophila have been shown to interfere with various host pathways and processes, but no L. pneumophila effector has ever been identified to be indispensable for lysosomal evasion. While most single effector mutants of L. pneumophila do not exhibit a defective phenotype within macrophages, we show that the MavE effector is essential for intracellular growth of L. pneumophila in human monocyte-derived macrophages (hMDMs) and amoebae and for intrapulmonary proliferation in mice. The mavE null mutant fails to remodel the LCV with endoplasmic reticulum (ER)-derived vesicles and is trafficked to the lysosomes where it is degraded, similar to formalin- killed bacteria. During infection of hMDMs, the MavE effector localizes to the poles of the LCV membrane. The crystal structure of MavE, resolved to 1.8 Å, reveals a C- terminal transmembrane helix, three copies of tyrosine- based sorting motifs, and an NPxY eukaryotic motif, which binds phosphotyrosine- binding domains present on signaling and adaptor eukaryotic proteins. Two point mutations within the NPxY motif result in attenuation of L. pneumophila in both hMDMs and amoeba. The substitution defects of P78 and D64 are associated with failure of vacuoles harboring the mutant to be remodeled by the ER and results in fusion of the vacuole to the lysosomes leading to bacterial degradation. Therefore, the MavE effector of L. pneumophila is indispensable for phagosome biogenesis and lysosomal evasion.IMPORTANCE Intracellular proliferation of Legionella pneumophila within a vacuole in human alveolar macrophages is essential for manifestation of Legionnaires' pneumonia. Intravacuolar growth of the pathogen is totally dependent on remodeling the L. pneumophila-containing vacuole (LCV) by the ER and on its evasion of the endosomal-lysosomal degradation pathway. The pathogen has evolved to inject ∼350 protein effectors into the host cell where they modulate various host processes, but no L. pneumophila effector has ever been identified to be indispensable for lysosomal evasion. We show that the MavE effector localizes to the poles of the LCV membrane and is essential for lysosomal evasion and intracellular growth of L. pneumophila and for intrapulmonary proliferation in mice. The crystal structure of MavE shows an NPxY eukaryotic motif essential for ER-mediated remodeling and lysosomal evasion by the LCV. Therefore, the MavE effector of L. pneumophila is indispensable for phagosome biogenesis and lysosomal evasion

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.

Mycobacterium tuberculosis is the main etiological agent of tuberculosis. The Bacillus Calmette–Guerin (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.

Legionella pneumophila invades protozoa with an “accidental” ability to cause pneumonia upon transmission to humans. To support its nutrition during intracellular residence, L. pneumophila relies on host amino acids as the main source of carbon and energy to feed the TCA cycle. Despite the apparent lack of a requirement for glucose for L. pneumophila growth in vitro and intracellularly, the organism contains multiple amylases, which hydrolyze polysaccharides into glucose monomers. Here we describe one predicted putative amylase, LamB, which is uniquely present only in L. pneumophila and L. steigerwaltii among the ~60 species of Legionella. Our data show that LamB has a strong amylase activity, which is abolished upon substitutions of amino acids that are conserved in the catalytic pocket of amylases. Loss of LamB or expression of catalytically-inactive variants of LamB results in a severe growth defect of L. pneumophila in Acanthamoeba polyphaga and human monocytes-derived macrophages. Importantly, the lamB null mutant is severely attenuated in intra-pulmonary proliferation in the mouse model and is defective in dissemination to the liver and spleen. Our data show an essential role for LamB in intracellular replication of L. pneumophila in amoeba and human macrophages and in virulence in vivo.

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 minutes 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.

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.

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.

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].

Francisella tularensis is a gram negative facultative intracellular bacterium that causes the zoonotic disease tularemia. Free-living amoebae, such as Acanthamoeba and Hartmanella, are environmental hosts of several intracellular pathogens. Epidemiology of F. tularensis in various parts of the world is associated with water-borne transmission, which includes mosquitoes and amoebae as the potential host reservoirs of the bacteria in water resources. In vitro studies showed intracellular replication of F. tularensis within A. castellanii cells. Whether amoeba is a biological reservoir for Francisella in the environment is not known. We used Hartmanella vermiformis as an amoebal model system to study the intracellular life of F. novicida. For the first time we show that F. novicida survives and replicates within H. vermiformis. The iglC mutant strain of F. novicida is defective for survival and replication not only within A. castellanii but also in H. vermiformis cells. In contrast to mammalian cells, where bacteria replicate in the cytosol, F. novicida resides and replicates within membrane-bound vacuoles within the trophozoites of H. vermiformis. In contrast to the transient residence of F. novicida within acidic vacuoles prior to escaping to the cytosol of mammalian cells, F. novicida does not reside transiently or permanently in an acidic compartment within H. vermiformis when examined 30 min after initiation of the infection. We conclude that F. tularensis does not replicate within acidified vacuoles and does not escape into the cytosol of H. vermiformis. The Francisella pathogenicity island locus iglC is essential for intra-vacuolar proliferation of F. novicida within H. vermiformis. Our data show a distinct intracellular lifestyle for F. novicida within H. vermiformis compared to mammalian cells.