The early phagosomal stage of Francisella tularensis determines optimal phagosomal escape and Francisella pathogenicity island protein expression

Francisella tularensis is an intracellular pathogen that can survive and replicate within macrophages. Following phagocytosis and transient interactions with the endocytic pathway, F. tularensis rapidly escapes from its original phagosome into the macrophage cytoplasm, where it eventually replicates. To examine the importance of the nascent phagosome for the Francisella intracellular cycle, we have characterized early trafficking events of the F. tularensis subsp. tularensis strain Schu S4 in a murine bone marrow-derived macrophage model. Here we show that early phagosomes containing Schu S4 transiently interact with early and late endosomes and become acidified before the onset of phagosomal disruption. Inhibition of endosomal acidification with the vacuolar ATPase inhibitor bafilomycin A1 or concanamycin A prior to infection significantly delayed but did not block phagosomal escape and cytosolic replication, indicating that maturation of the early Francisella-containing phagosome (FCP) is important for optimal phagosomal escape and subsequent intracellular growth. Further, Francisella pathogenicity island (FPI) protein expression was induced during early intracellular trafficking events. Although inhibition of endosomal acidification mimicked the early phagosomal escape defects caused by mutation of the FPI-encoded IglCD proteins, it did not inhibit the intracellular induction of FPI proteins, demonstrating that this response is independent of phagosomal pH. Altogether, these results demonstrate that early phagosomal maturation is required for optimal phagosomal escape and that the early FCP provides cues other than intravacuolar pH that determine intracellular induction of FPI proteins.     

Characterization of the receptor-ligand pathways important for entry and survival of Francisella tularensis in human macrophages

Inhalational pneumonic tularemia, caused by Francisella tularensis, is lethal in humans. F. tularensis is phagocytosed by macrophages followed by escape from phagosomes into the cytoplasm. Little is known of the phagocytic mechanisms for Francisella, particularly as they relate to the lung and alveolar macrophages. Here we examined receptors on primary human monocytes and macrophages which mediate the phagocytosis and intracellular survival of F. novicida. F. novicida association with monocyte-derived macrophages (MDM) was greater than with monocytes. Bacteria were readily ingested, as shown by electron microscopy. Bacterial association was significantly increased in fresh serum and only partially decreased in heat-inactivated serum. A role for both complement receptor 3 (CR3) and Fcγ receptors in uptake was supported by studies using a CR3-expressing cell line and by down-modulation of Fcγ receptors on MDM, respectively. Consistent with Fcγ receptor involvement, antibody in nonimmune human serum was detected on the surface of Francisella. In the absence of serum opsonins, competitive inhibition of mannose receptor (MR) activity on MDM with mannan decreased the association of F. novicida and opsonization of F. novicida with lung collectin surfactant protein A (SP-A) increased bacterial association and intracellular survival. This study demonstrates that human macrophages phagocytose more Francisella than monocytes with contributions from CR3, Fcγ receptors, the MR, and SP-A present in lung alveoli.     

Importance of Branched-Chain Amino Acid Utilization in Francisella Intracellular Adaptation

Intracellular bacterial pathogens have adapted their metabolism to optimally utilize the nutrients available in infected host cells. We recently reported the identification of an asparagine transporter required specifically for cytosolic multiplication of Francisella. In the present work, we characterized a new member of the major super family (MSF) of transporters, involved in isoleucine uptake. We show that this transporter (here designated IleP) plays a critical role in intracellular metabolic adaptation of Francisella. Inactivation of IleP severely impaired intracellular F. tularensis subsp. novicida multiplication in all cell types tested and reduced bacterial virulence in the mouse model. To further establish the importance of the ileP gene in F. tularensis pathogenesis, we constructed a chromosomal deletion mutant of ileP (ΔFTL_1803) in the F. tularensis subsp. holarctica live vaccine strain (LVS). Inactivation of IleP in the F. tularensis LVS provoked comparable intracellular growth defects, confirming the critical role of this transporter in isoleucine uptake. The data presented establish, for the first time, the importance of isoleucine utilization for efficient phagosomal escape and cytosolic multiplication of Francisella and suggest that virulent F. tularensis subspecies have lost their branched-chain amino acid biosynthetic pathways and rely exclusively on dedicated uptake systems. This loss of function is likely to reflect an evolution toward a predominantly intracellular life style of the pathogen. Amino acid transporters should be thus considered major players in the adaptation of intracellular pathogens.     

RipA, a cytoplasmic membrane protein conserved among Francisella species, is required for intracellular survival

Francisella tularensis is a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of the F. tularensis live vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequenced Francisella species yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele in trans. Based on the deletion mutant phenotype, FTL_1914 was termed ripA (required for intracellular proliferation, factor A). Following uptake by J774.A1 cells, F. tularensis LVS ΔripA colocalized with LAMP-1 then escaped the phagosome at the same rate and frequency as wild-type LVS-infected cells. Electron micrographs of the F. tularensis LVS ΔripA mutant demonstrated the reentry of the mutant bacteria into double membrane vacuoles characteristic of autophagosomes in a process that was not dependent on replication. The F. tularensis LVS ΔripA mutant was significantly impaired in its ability to persist in the lung and in its capacity to disseminate and colonize the liver and spleen in a mouse model of pulmonary tularemia. The RipA protein was expressed during growth in laboratory media and localized to the cytoplasmic membrane. Thus, RipA is a cytoplasmic membrane protein conserved among Francisella species that is required for intracellular replication within the host cell cytoplasm as well as disease progression, dissemination, and virulence.     

Identification of Early Interactions between Francisella and the Host

The adaptive immune response to Francisella tularensis is dependent on the route of inoculation. Intradermal inoculation with the F. tularensis live vaccine strain (LVS) results in a robust Th1 response in the lungs, whereas intranasal inoculation produces fewer Th1 cells and instead many Th17 cells. Interestingly, bacterial loads in the lungs are similar early after inoculation by these two routes. We hypothesize that the adaptive immune response is influenced by local events in the lungs, such as the type of cells that are first infected with Francisella. Using fluorescence-activated cell sorting, we identified alveolar macrophages as the first cell type infected in the lungs of mice intranasally inoculated with F. novicida U112, LVS, or F. tularensis Schu S4. Following bacterial dissemination from the skin to the lung, interstitial macrophages or neutrophils are infected. Overall, we identified the early interactions between Francisella and the host following two different routes of inoculation.     

Interleukin-17 Protects against the Francisella tularensis Live Vaccine Strain but Not against a Virulent F. tularensis Type A Strain

Francisella tularensis is a highly infectious intracellular bacterium that causes the zoonotic infection tularemia. While much literature exists on the host response to F. tularensis infection, the vast majority of work has been conducted using attenuated strains of Francisella that do not cause disease in humans. However, emerging data indicate that the protective immune response against attenuated F. tularensis versus F. tularensis type A differs. Several groups have recently reported that interleukin-17 (IL-17) confers protection against the live vaccine strain (LVS) of Francisella. While we too have found that IL-17Rα^−/− mice are more susceptible to F. tularensis LVS infection, our studies, using a virulent type A strain of F. tularensis (SchuS4), indicate that IL-17Rα^−/− mice display organ burdens and pulmonary gamma interferon (IFN-γ) responses similar to those of wild-type mice following infection. In addition, oral LVS vaccination conferred equivalent protection against pulmonary challenge with SchuS4 in both IL-17Rα^−/− and wild-type mice. While IFN-γ was found to be critically important for survival in a convalescent model of SchuS4 infection, IL-17 neutralization from either wild-type or IFN-γ^−/− mice had no effect on morbidity or mortality in this model. IL-17 protein levels were also higher in the lungs of mice infected with the LVS rather than F. tularensis type A, while IL-23p19 mRNA expression was found to be caspase-1 dependent in macrophages infected with LVS but not SchuS4. Collectively, these results demonstrate that IL-17 is dispensable for host immunity to type A F. tularensis infection, and that induced and protective immunity differs between attenuated and virulent strains of F. tularensis.     

Francisella tularensis LVS grown in macrophages has reduced ability to stimulate the secretion of inflammatory cytokines by macrophages in vitro

The virulence of Francisella tularensis LVS is determined in part by its ability to invade and replicate within macrophages and stimulate the production of inflammatory cytokines. The present study determined the effects of growing F. tularensis in macrophages on its ability to stimulate cytokine secretion by macrophages. F. tularensis grown in Mueller–Hinton broth (FtB) stimulated the secretion of large amounts of TNF-α, IL-12p40, IL-6 and MCP-1/CCL2 when incubated with macrophages overnight. In contrast, F. tularensis released from infected macrophages (FtMac) stimulated very little secretion of these cytokines by primary cultures of murine peritoneal macrophages, human monocytes or macrophage cell lines. Stimulation of nitric oxide production by FtMac was also less than that elicited by FtB. FtMac killed with gentamicin or paraformaldehyde also stimulated low levels of cytokine secretion. FtMac recovered the ability to stimulate cytokine secretion after overnight culture in broth. Infection of macrophages with FtMac inhibited the cytokine response to subsequent stimulation with LPS from Escherichia coli but did not affect Fcγ receptor-mediated phagocytosis. FtMac were ingested by macrophages at about half the rate of FtB, however, this did not account for the lower cytokine secretion. FtMac and FtB replicated at similar rates within macrophages. Finally, Mice infected with FtMac had a higher mortality rate than those infected with FtB. These results reveal that growth in macrophages causes a reversible phenotypic change in F. tularensis that is associated with decreased stimulation of cytokine secretion, inhibition of LPS-stimulated secretion of inflammatory cytokines by macrophages and increased lethality in mice.     

Activation of B cell apoptotic pathways in the course of Francisella tularensis infection

Francisella tularensis is a facultative intracellular, gram-negative bacterium that induces apoptosis in macrophages and B cells. Here we show apoptotic pathways that are activated in the Ramos human B cell line in the course of F. tularensis infection. Live bacteria F. tularensis FSC200 activate caspases 8, 9 and 3, as well as Bid; release cytochrome c and apoptosis-inducing factor from mitochondria; and induce depolarization of mitochondrial membrane potential in the Ramos cell line, thus leading these cells to apoptosis. Unlike live bacteria, killed F. tularensis FSC200 bacteria activated only caspase 3, and did not cause apoptosis of Ramos cells as measured by annexin V. Killed bacteria also caused accumulation of anti-apoptotic protein Bclx_L in mitochondrial membranes. Thus, live F. tularensis activates both caspase pathways (receptor-mediated and intrinsic) as well as caspase-independent mitochondrial death.     

Molecular method for discrimination between Francisella tularensis and Francisella-like endosymbionts

Environmental studies on the distribution of Francisella spp. are hampered by the frequency of Francisella-like endosymbionts that can produce a misleading positive result. A new, efficient molecular method for detection of Francisella tularensis and its discrimination from Francisella-like endosymbionts, as well as two variants associated with human disease (unusual F. tularensis strain FnSp1 and F. tularensis subsp. novicida-like strain 3523), is described. The method is highly specific and sensitive, detecting up to one plasmid copy or 10 genome equivalents.     

Identifying Francisella tularensis Genes Required for Growth in Host Cells

Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The ΔFTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The ΔFTT_0924 mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining peptidoglycan integrity and virulence.     

Disruption of Francisella tularensis Schu S4 iglI,iglJ, and pdpC Genes Results in Attenuation for Growth in Human Macrophages and In Vivo Virulence in Mice and Reveals a Unique Phenotype for pdpC

Francisella tularensis is a facultative intracellular bacterial pathogen and the causative agent of tularemia. After infection of macrophages, the organism escapes from its phagosome and replicates to high density in the cytosol, but the bacterial factors required for these aspects of virulence are incompletely defined. Here, we describe the isolation and characterization of Francisella tularensis subsp. tularensis strain Schu S4 mutants that lack functional iglI, iglJ, or pdpC, three genes of the Francisella pathogenicity island. Our data demonstrate that these mutants were defective for replication in primary human monocyte-derived macrophages and murine J774 cells yet exhibited two distinct phenotypes. The iglI and iglJ mutants were similar to one another, exhibited profound defects in phagosome escape and intracellular growth, and appeared to be trapped in cathepsin D-positive phagolysosomes. Conversely, the pdpC mutant avoided trafficking to lysosomes, phagosome escape was diminished but not ablated, and these organisms replicated in a small subset of infected macrophages. The phenotype of each mutant strain was reversed by trans complementation. In vivo virulence was assessed by intranasal infection of BALB/c mice. The mutants appeared avirulent, as all mice survived infection with 10⁸ CFU iglJ- or pdpC-deficient bacteria. Nevertheless, the pdpC mutant disseminated to the liver and spleen before being eliminated, whereas the iglJ mutant did not. Taken together, our data demonstrate that the pathogenicity island genes tested are essential for F. tularensis Schu S4 virulence and further suggest that pdpC may play a unique role in this process, as indicated by its distinct intermediate phenotype.     

Identification of T-cell epitopes in Francisella tularensis using an ordered protein array of serological targets

Francisella tularensis is a Gram‐negative intracellular bacterium that is the causative agent of tularaemia. Concerns regarding its use as a bioterrorism agent have led to a renewed interest in the biology of infection, host response and pathogenesis. A robust T‐cell response is critical to confer protection against F. tularensis. However, characterization of the cellular immune response has been hindered by the paucity of tools to examine the anti‐Francisella immune response at the molecular level. We set out to combine recent advances of genomics with solid‐phase antigen delivery coupled with a T‐cell functional assay to identify T‐cell epitopes. A subset of clones, encoding serological targets, was selected from an F. tularensis SchuS4 ordered genomic library and subcloned into a bacterial expression vector to test the feasibility of this approach. Proteins were expressed and purified individually employing the BioRobot 3000 in a semi‐automated purification method. The purified proteins were coupled to beads, delivered to antigen‐presenting cells for processing, and screened with Francisella‐specific T‐cell hybridomas of unknown specificity. We identified cellular reactivity against the pathogenicity protein IglB, and the chaperone proteins GroEL and DnaK. Further analyses using genetic deletions and synthetic peptides were performed to identify the minimal peptide epitopes. Priming with the peptide epitopes before infection with F. tularensis LVS increased the frequency of antigen‐specific CD4 T cells as assessed by intracellular interferon‐γ staining. These results illustrate the feasibility of screening an arrayed protein library that should be applicable to a variety of pathogens.     

IglE Is an Outer Membrane-Associated Lipoprotein Essential for Intracellular Survival and Murine Virulence of Type A Francisella tularensis

IglE is a small, hypothetical protein encoded by the duplicated Francisella pathogenicity island (FPI). Inactivation of both copies of iglE rendered Francisella tularensis subsp. tularensis Schu S4 avirulent and incapable of intracellular replication, owing to an inability to escape the phagosome. This defect was fully reversed following single-copy expression of iglE in trans from attTn7 under the control of the Francisella rpsL promoter, thereby establishing that the loss of iglE, and not polar effects on downstream vgrG gene expression, was responsible for the defect. IglE is exported to the Francisella outer membrane as an ∼13.9-kDa lipoprotein, determined on the basis of a combination of selective Triton X-114 solubilization, radiolabeling with [³H]palmitic acid, and sucrose density gradient membrane partitioning studies. Lastly, a genetic screen using the iglE-null live vaccine strain resulted in the identification of key regions in the carboxyl terminus of IglE that are required for intracellular replication of Francisella tularensis in J774A.1 macrophages. Thus, IglE is essential for Francisella tularensis virulence. Our data support a model that likely includes protein-protein interactions at or near the bacterial cell surface that are unknown at present.     

Infected-host-cell repertoire and cellular response in the lung following inhalation of Francisella tularensis Schu S4, LVS, or U112

Francisella tularensis causes systemic disease in humans and other mammals, with high morbidity and mortality associated with inhalation-acquired infection. F. tularensis is a facultative intracellular pathogen, but the scope and significance of cell types infected during disease is unknown. Using flow cytometry, we identified and quantified infected-cell types and assessed the impact of infection on cell populations following inhalation of F. tularensis strains U112, LVS, and Schu S4. Initially, alveolar macrophages comprised over 70% of Schu S4- and LVS-infected cells, whereas approximately 51% and 27% of U112-infected cells were alveolar macrophages and neutrophils, respectively. After 3 days, roughly half of Schu S4- and LVS- and nearly 80% of U112-infected cells were neutrophils. All strains infected CD11b^high macrophages, dendritic cells, monocytes, and alveolar type II cells throughout infection. Macrophage, monocyte, and dendritic-cell populations were reduced during U112 infection but not Schu S4 or LVS infection. These results demonstrate directly that F. tularensis is a promiscuous intracellular pathogen in the lung that invades and replicates within cell types ranging from migratory immune cells to structural tissue cells. However, the proportions of cell types infected and the cellular immune response evoked by the human pathogenic strain Schu S4 differ from those of the human avirulent U112.     

Combined deletion of four Francisella novicida acid phosphatases attenuates virulence and macrophage vacuolar escape

Francisella tularensis is a facultative intracellular pathogen and the etiologic agent of tularemia. It is capable of escape from macrophage phagosomes and replicates in the host cell cytosol. Bacterial acid phosphatases are thought to play a major role in the virulence and intracellular survival of a number of intracellular pathogens. The goal of this study was to delete the four primary acid phosphatases (Acps) from Francisella novicida and examine the interactions of mutant strains with macrophages, as well as the virulence of these strains in mice. We constructed F. novicida mutants with various combinations of acp deletions and showed that loss of the four Acps (AcpA, AcpB, AcpC, and histidine acid phosphatase [Hap]) in an F. novicida strain (ΔABCH) resulted in a 90% reduction in acid phosphatase activity. The ΔABCH mutant was defective for survival/growth within human and murine macrophage cell lines and was unable to escape from phagosome vacuoles. With accumulation of Acp deletions, a progressive loss of virulence in the mouse model was observed. The ΔABCH strain was dramatically attenuated and was an effective single-dose vaccine against homologous challenge. Furthermore, both acpA and hap were induced when the bacteria were within host macrophages. Thus, the Francisella acid phosphatases cumulatively play an important role in intracellular trafficking and virulence.     

Importance of PdpC,IglC, IglI,and IglG for Modulation of a Host Cell Death Pathway Induced by Francisella tularensis

Modulation of host cell death pathways appears to be a prerequisite for the successful lifestyles of many intracellular pathogens. The facultative intracellular bacterium Francisella tularensis is highly pathogenic, and effective proliferation in the macrophage cytosol leading to host cell death is a requirement for its virulence. To better understand the prerequisites of this cell death, macrophages were infected with the F. tularensis live vaccine strain (LVS), and the effects were compared to those resulting from infections with deletion mutants lacking expression of either of the pdpC, iglC, iglG, or iglI genes, which encode components of the Francisella pathogenicity island (FPI), a type VI secretion system. Within 12 h, a majority of the J774 cells infected with the LVS strain showed production of mitochondrial superoxide and, after 24 h, marked signs of mitochondrial damage, caspase-9 and caspase-3 activation, phosphatidylserine expression, nucleosome formation, and membrane leakage. In contrast, neither of these events occurred after infection with the ΔiglI or ΔiglC mutants, although the former strain replicated. The ΔiglG mutant replicated effectively but induced only marginal cytopathogenic effects after 24 h and intermediate effects after 48 h. In contrast, the ΔpdpC mutant showed no replication but induced marked mitochondrial superoxide production and mitochondrial damage, caspase-3 activation, nucleosome formation, and phosphatidylserine expression, although the effects were delayed compared to those obtained with LVS. The unique phenotypes of the mutants provide insights regarding the roles of individual FPI components for the modulation of the cytopathogenic effects resulting from the F. tularensis infection.     

MglA and Igl proteins contribute to the modulation of Francisella tularensis live vaccine strain-containing phagosomes in murine macrophages

The Francisella tularensis live vaccine strain (LVS), in contrast to its iglC mutant, replicates in the cytoplasm of macrophages. We studied the outcome of infection of the murine macrophagelike cell line J774A.1 with LVS and with iglC, iglD, and mglA mutants, the latter of which is deficient in a global regulator. Compared to LVS, all of the mutants showed impaired intracellular replication up to 72 h, and the number of the mglA mutant bacteria even decreased. Colocalization with LAMP-1 was significantly increased for all mutants compared to LVS, indicating an impaired ability to escape into the cytoplasm. A lysosomal acidity-dependent dye accumulated in approximately 40% of the vacuoles containing mutant bacteria but not at all in vacuoles containing LVS. Preactivation of the macrophages with gamma interferon inhibited the intracellular growth of all strains and significantly increased acidification of phagosomes containing the mutants, but it only slightly increased the LAMP-1 colocalization. The intracellular replication and phagosomal escape of the iglC and iglD mutants were restored by complementation in trans. In conclusion, the IglC, IglD, and MglA proteins each directly or indirectly critically contribute to the virulence of F. tularensis LVS, including its intracellular replication, cytoplasmic escape, and inhibition of acidification of the phagosomes.     

Directed Screen of Francisella novicida Virulence Determinants Using Drosophila melanogaster

Francisella tularensis is a highly virulent, facultative intracellular human pathogen whose virulence mechanisms are not well understood. Occasional outbreaks of tularemia and the potential use of F. tularensis as a bioterrorist agent warrant better knowledge about the pathogenicity of this bacterium. Thus far, genome-wide in vivo screens for virulence factors have been performed in mice, all however restricted by the necessity to apply competition-based, negative-selection assays. We wanted to individually evaluate putative virulence determinants suggested by such assays and performed directed screening of 249 F. novicida transposon insertion mutants by using survival of infected fruit flies as a measure of bacterial virulence. Some 20% of the genes tested were required for normal virulence in flies; most of these had not previously been investigated in detail in vitro or in vivo. We further characterized their involvement in bacterial proliferation and pathogenicity in flies and in mouse macrophages. Hierarchical cluster analysis of mutant phenotypes indicated a functional linkage between clustered genes. One cluster grouped all but four genes of the Francisella pathogenicity island and other loci required for intracellular survival. We also identified genes involved in adaptation to oxidative stress and genes which might induce host energy wasting. Several genes related to type IV pilus formation demonstrated hypervirulent mutant phenotypes. Collectively, the data demonstrate that the bacteria in part use similar virulence mechanisms in mammals as in Drosophila melanogaster but that a considerable proportion of the virulence factors active in mammals are dispensable for pathogenicity in the insect model.Francisella tularensis is the causative agent of tularemia, a zoonotic disease affecting a wide variety of small vertebrates as well as humans (49). The severity and the clinical manifestations of the disease are highly dependent on the infecting strain and the route of entry. If inhaled, as few as 10 bacteria can cause infection in humans, and if untreated, the mortality rate can reach 60% (45). To date, two subspecies that cause disease in humans, F. tularensis subspecies holarctica and F. tularensis subspecies tularensis, have been identified. A closely related species, F. novicida, is an environmental pathogen and appears not to affect healthy humans, since F. novicida infections have been reported almost exclusively in immunocompromised individuals (8, 19, 58), but it has been recognized as relevant when Francisella virulence in various mouse infection models is investigated. Genome comparisons revealed high sequence similarities between the F. novicida isolate U112 and the clinically important F. tularensis subspecies tularensis strain SCHU S4. The former represents the evolutionarily oldest and most complete Francisella genome, supporting good metabolic competence, while human-pathogenic strains, in adaptation to an intracellular niche, have lost many genes to genetic drift and are much more fastidious (26, 43).The only tularemia treatment to date relies on antibiotics. To enable development of vaccines and new antimicrobial drugs, it is vital to understand the molecular mechanisms behind the interaction of this pathogen with humans. F. tularensis escapes from the host cell phagosome and propagates in the cytosol (16). Multiplication results in cell death and release of bacteria (25), allowing them to spread to regional lymph nodes and to colonize spleen, liver, and lung (52). A substantial proportion of the bacterial burden can persist extracellularly in the bloodstream (14, 59).Despite knowledge about the in vivo life cycle, genome sequence data, and techniques for mutant generation, we still know little about specific virulence determinants of F. tularensis. Factors that are known to play a role are involved in lipopolysaccharide biosynthesis or intracellular survival. A focus of attention has been a genomic region called the Francisella pathogenicity island (FPI), which is required for escape from the phagosome and proliferation inside the cytosol and which encodes a putative type VI secretion system (reviewed in references 2, 13, and 35).Lately, libraries of transposon insertion mutants of different Francisella reference strains were used to screen for virulence factors in various mammalian in vitro and in vivo infection models (23, 31, 38, 51, 53, 57). In vivo screens in mice applied a competition-based, negative-selection strategy to identify bacterial mutants that cannot survive in and/or colonize a target organ (23, 51, 57). Results from these studies suggested a large number of genes to be involved in Francisella pathogenicity. While this strategy is a sensitive and efficient way to screen the whole bacterial genome, it probably overestimates the number of virulence genes and their importance as well as providing limited information as to the specific roles of individual genes in pathogenesis.Thus, far, the mouse has been the preferred model host for in vivo studies of Francisella. Recently, however, analysis of various human pathogens in model organisms like Caenorhabditis elegans or Drosophila melanogaster have demonstrated that bacteria to a large extent rely on the same virulence strategies in invertebrates as in humans (24). Because of their simplicity and the genetic tools available, nonmammalian models offer a unique opportunity to unravel the basis of host-pathogen interactions in great detail. Our previous work, in which we introduced D. melanogaster as a model host for Francisella infections, suggested that the fruit fly might be valuable for the identification and characterization of virulence determinants (56). In addition, blood-feeding arthropods, like ticks, mosquitoes, and biting flies, have long been acknowledged as vectors of tularemia (34, 37), implying that the bacterium has evolved strategies for persistence and replication in such organisms. Drosophila melanogaster might not represent a natural host for Francisella tularensis, but it is a highly relevant model for immune mechanisms and basic physiology in arthropods.Here we individually analyzed nearly 250 genes that had previously been suggested as candidate virulence determinants by negative-selection screens in mice in order to confirm their importance for pathogenicity in a robust infection model. By using survival of infected flies as a measure of bacterial virulence, we identified 49 genes as being required for normal virulence in flies. These genes were further investigated for their role in bacterial proliferation in flies and in mouse macrophage-like cells. All mutant phenotypes were analyzed by hierarchical cluster analysis to provide new insights into functional relationships among the corresponding genes. Our collected data and comparison of bacterial mutants in two model systems also allowed for an evaluation of D. melanogaster as a screening model.     

Francisella tularensis Phagosomal Escape Does Not Require Acidification of the Phagosome

Following uptake, Francisella tularensis enters a phagosome that acquires limited amounts of lysosome-associated membrane glycoproteins and does not acquire cathepsin D or markers of secondary lysosomes. With additional time after uptake, F. tularensis disrupts its phagosomal membrane and escapes into the cytoplasm. To assess the role of phagosome acidification in phagosome escape, we followed acidification using the vital stain LysoTracker red and acquisition of the proton vacuolar ATPase (vATPase) using immunofluorescence within the first 3 h after uptake of live or killed F. tularensis subsp. holarctica live vaccine strain (LVS) by human macrophages. Whereas 90% of the phagosomes containing killed LVS stained intensely for the vATPase and were acidified, only 20 to 30% of phagosomes containing live LVS stained intensely for the vATPase and were acidified. To determine whether transient acidification might be required for phagosome escape, we assessed the impact on phagosome permeabilization of the proton pump inhibitor bafilomycin A. Using electron microscopy and an adenylate cyclase reporter system, we found that bafilomycin A did not prevent phagosomal permeabilization by F. tularensis LVS or virulent type A strains (F. tularensis subsp. tularensis strain Schu S4 and a recent clinical isolate) or by “F. tularensis subsp. novicida,” indicating that F. tularensis disrupts its phagosomal membrane by a mechanism that does not require acidification.Francisella tularensis is a gram-negative facultative intracellular bacterium that causes a zoonosis in animals and a potentially fatal infection, tularemia, in humans. F. tularensis consists of four subspecies, F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, F. tularensis subsp. mediasiatica and “F. tularensis subsp. novicida,” whose geographic distributions and virulence in humans differ (12, 25). F. tularensis subsp. tularensis (type A), found almost exclusively in North America, is highly virulent for humans. As few as 10 organisms received subcutaneously or 25 organisms received by inhalation can lead to a severe infection (32, 33). F. tularensis subsp. holarctica (type B, found in North America and in Europe) and F. tularensis subsp. mediasiatica (found in Asia) are less virulent. F. tularensis subsp. novicida, found in North America and Australia, is virulent in mice and has occasionally been reported to cause a mild disease, compared with type A infections, in humans (38). Because of its high infectivity and capacity to cause severe morbidity and mortality, F. tularensis subsp. tularensis is considered a potential agent of bioterrorism and is classified as a category A select agent.In animal models of tularemia, macrophages are important host cells for F. tularensis, and the virulence of the bacterium correlates with its capacity to grow in macrophages (2, 20). We have shown previously that efficient uptake of F. tularensis subsp. tularensis and F. tularensis subsp. holarctica live vaccine strain (LVS) by human macrophages requires complement and that it is mediated by a unique process involving spacious, asymmetric pseudopod loops (10). The mannose receptor (34) and class A scavenger receptors (26) have also been reported to play a role in uptake of F. tularensis LVS. We have demonstrated that following uptake, the bacterium enters a membrane-bound vacuole that acquires limited amounts of endosomal markers, including limited amounts of the late endosomal-lysosomal markers CD63, LAMP1, and LAMP2, but that the vacuole does not acquire the acid hydrolase cathepsin D, does not fuse with lysosomes, and is only minimally acidified to a pH of 6.7 at 3 h postinfection (11). With additional time after uptake, F. tularensis disrupts the phagosomal membrane and the bacterium escapes and replicates in the host cell cytosol (9, 11, 16). Celli and coworkers have studied the interaction of mouse bone marrow-derived macrophages with F. tularensis LVS (6) and F. tularensis Schu S4 (7) and also reported a transient interaction with the host endocytic pathway prior to escape with a more rapid kinetic profile than we have observed in human monocyte-derived macrophages (MDM). In addition, Chercoun et al. (6) have reported that at late times after infection (20 h) in mouse macrophages, a large proportion of F. tularensis cells enter an autophagosomal compartment. The Francisella pathogenicity island has been shown to be essential for the altered intracellular trafficking and escape of F. tularensis subsp. holarctica LVS (22) and F. tularensis subsp. novicida (31) into the cytoplasm.Some degree of acidification has been shown to be required for the escape of certain intracellular pathogens that replicate in the cytosol. For example, acidification of the vacuole occupied by Listeria monocytogenes is required for activation of listeriolysin O for permeablization of the vacuole (1), and acidification of either early or late endosomes is required for pH-dependent changes in adenoviral proteins to mediate the translocation of adenovirus into the host cell cytoplasm (23). While we have reported previously that the F. tularensis phagosome is only minimally acidified to a pH of 6.7 at 3 h postinfection, this finding does not preclude the possibility that some degree of acidification, even transient acidification, might be required for the bacterium to disrupt its phagosome and escape into the cytoplasm. Indeed, Santic et al. recently reported that nearly 80 to 85% of F. tularensis subsp. novicida phagosomes are acidified at 15 to 30 min postinfection in human MDM and that inhibition of acidification with bafilomycin A completely blocks escape (30). In contrast to these results for human macrophages with F. tularensis subsp. novicida, Chong et al. (7) have recently reported that F. tularensis Schu S4 phagosomes in mouse bone marrow-derived macrophages are transiently acidified and that inhibition of acidification delays, but does not prevent, phagosome disruption. To explore the importance of phagosomal pH on subsequent intracellular trafficking events for F. tularensis in human macrophages, we have examined the time course of colocalization of F. tularensis with the proton vacuolar ATPase (vATPase) and with a vital stain for acidified compartments, and we have examined the effect of inhibitors of acidification on phagosomal disruption.