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.     

Novel Catanionic Surfactant Vesicle Vaccines Protect against Francisella tularensis LVS and Confer Significant Partial Protection against F. tularensis Schu S4 Strain

Francisella tularensis is a Gram-negative immune-evasive coccobacillus that causes tularemia in humans and animals. A safe and efficacious vaccine that is protective against multiple F. tularensis strains has yet to be developed. In this study, we tested a novel vaccine approach using artificial pathogens, synthetic nanoparticles made from catanionic surfactant vesicles that are functionalized by the incorporation of either F. tularensis type B live vaccine strain (F. tularensis LVS [LVS-V]) or F. tularensis type A Schu S4 strain (F. tularensis Schu S4 [Schu S4-V]) components. The immunization of C57BL/6 mice with “bare” vesicles, which did not express F. tularensis components, partially protected against F. tularensis LVS, presumably through activation of the innate immune response, and yet it failed to protect against the F. tularensis Schu S4 strain. In contrast, immunization with LVS-V fully protected mice against intraperitoneal (i.p.) F. tularensis LVS challenge, while immunization of mice with either LVS-V or Schu S4-V partially protected C57BL/6 mice against an intranasal (i.n.) F. tularensis Schu S4 challenge and significantly increased the mean time to death for nonsurvivors, particularly following the i.n. and heterologous (i.e., i.p./i.n.) routes of immunization. LVS-V immunization, but not immunization with empty vesicles, elicited high levels of IgG against nonlipopolysaccharide (non-LPS) epitopes that were increased after F. tularensis LVS challenge and significantly increased early cytokine production. Antisera from LVS-V-immunized mice conferred passive protection against challenge with F. tularensis LVS. Together, these data indicate that functionalized catanionic surfactant vesicles represent an important and novel tool for the development of a safe and effective F. tularensis subunit vaccine and may be applicable for use with other pathogens.     

Impact of Francisella tularensis pilin homologs on pilus formation and virulence

Francisella tularensis is a facultative intracellular bacterium and the causative agent of tularemia. Virulence factors for this bacterium, particularly those that facilitate host cell interaction, remain largely uncharacterized. However, genes homologous to those involved in type IV pilus structure and assembly, including six genes encoding putative major pilin subunit proteins, are present in the genome of the highly virulent Schu S4 strain. To analyze the roles of three putative pilin genes in pili structure and function we constructed individual pilE4, pilE5, and pilE6 deletion mutants in both the F. tularensis tularensis strain Schu S4 and the Live Vaccine Strain (LVS), an attenuated derivative strain of F. tularensis holarctica. Transmission electron microscopy (TEM) of Schu S4 and LVS wild-type and deletion strains confirmed that pilE4 was essential for the expression of type IV pilus-like fibers by both subspecies. By the same method, pilE5 and pilE6 were dispensable for pilus production. In vitro adherence assays with J774A.1 cells revealed that LVS pilE4, pilE5, and pilE6 deletion mutants displayed increased attachment compared to wild-type LVS. However, in the Schu S4 background, similar deletion mutants displayed adherence levels similar to wild-type. In vivo, LVS pilE5 and pilE6 deletion mutants were significantly attenuated compared to wild-type LVS by intradermal and subcutaneous murine infection, while no Schu S4 deletion mutant was significantly attenuated compared to wild-type Schu S4. While pilE4 was essential for fiber expression on both Schu S4 and LVS, neither its protein product nor the assembled fibers contributed significantly to virulence in mice. Absent a role in pilus formation, we speculate PilE5 and PilE6 are pseudopilin homologs that comprise, or are associated with, a novel type II-related secretion system in Schu S4 and LVS.     

Expansion of Vγ9Vδ2 T Cells Is Triggered by Francisella tularensis-Derived Phosphoantigens in Tularemia but Not after Tularemia Vaccination

Tularemia is a disease caused by the facultative intracellular bacterium Francisella tularensis. Here we demonstrate that during the first weeks of infection, a significant increase in levels of Vγ9Vδ2 cells occurred in peripheral blood: in 13 patients analyzed 7 to 18 days after the onset of disease, these lymphocytes represented, on average, 30.5% of CD3⁺ cells and nearly 100% of γδ⁺ T cells. By contrast, after vaccination with the live vaccine strain (LVS) of F. tularensis, only a minor increase occurred. Eleven days after vaccination, γδ T cells represented an average of 6.7% and Vγ9Vδ2 cells represented an average of 5.3% of T cells, as in control subjects. Since derivatives of nonpeptidic pyrophosphorylated molecules, referred to as phosphoantigens, are powerful stimuli for Vγ9Vδ2 cells, this observation prompted an investigation of phosphoantigens in F. tularensis strains. The F. tularensis phosphoantigens triggered in vitro a proliferative response of human Vγ9Vδ2 peripheral blood leukocytes as well as a cytotoxic response and tumor necrosis factor release from a Vγ9Vδ2 T-cell clone. Quantitatively similar phosphoantigenic activity was detected in acellular extracts from two clinical isolates (FSC171 and Schu) and from LVS. Taken together, the chemical nature of the stimulus from the clinical isolates and the significant increase in levels of Vγ9Vδ2 cells in peripheral blood of tularemia patients indicate that phosphoantigens produced by virulent strains of F. tularensis trigger in vivo expansion of γδ T cells in tularemia.     

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.     

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.     

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.     

Francisella tularensis ΔpyrF Mutants Show that Replication in Nonmacrophages Is Sufficient for Pathogenesis In Vivo

The pathogenesis of Francisella tularensis has been associated with this bacterium''s ability to replicate within macrophages. F. tularensis can also invade and replicate in a variety of nonphagocytic host cells, including lung and kidney epithelial cells and hepatocytes. As uracil biosynthesis is a central metabolic pathway usually necessary for pathogens, we characterized ΔpyrF mutants of both F. tularensis LVS and Schu S4 to investigate the role of these mutants in intracellular growth. As expected, these mutant strains were deficient in de novo pyrimidine biosynthesis and were resistant to 5-fluoroorotic acid, which is converted to a toxic product by functional PyrF. The F. tularensis ΔpyrF mutants could not replicate in primary human macrophages. The inability to replicate in macrophages suggested that the F. tularensis ΔpyrF strains would be attenuated in animal infection models. Surprisingly, these mutants retained virulence during infection of chicken embryos and in the murine model of pneumonic tularemia. We hypothesized that the F. tularensis ΔpyrF strains may replicate in cells other than macrophages to account for their virulence. In support of this, F. tularensis ΔpyrF mutants replicated in HEK-293 cells and normal human fibroblasts in vitro. Moreover, immunofluorescence microscopy showed abundant staining of wild-type and mutant bacteria in nonmacrophage cells in the lungs of infected mice. These findings indicate that replication in nonmacrophages contributes to the pathogenesis of F. tularensis.Francisella tularensis causes the acute illness known as tularemia and is classified by the Centers for Disease Control and Prevention as a category A biodefense agent. This organism is highly infectious, as a single bacterium can lead to disease that may be fatal if untreated (16, 39, 53). The virulence of F. tularensis has been associated with this organism''s ability to replicate within phagocytic cells of the innate immune system, such as macrophages (4). In the murine model of respiratory tularemia, both airway macrophages and dendritic cells are infected within 1 h following inhalation of F. tularensis (7). As this disease progresses, macrophage numbers decline (26, 63), likely due to the induction of caspase-3-dependent apoptosis induced by the virulent type A F. tularensis strain (63). During interactions with host macrophages, F. tularensis can block the activation of these cells, as evidenced by the inhibition of proinflammatory cytokine production (9, 60, 61). In addition to macrophages and dendritic cells, F. tularensis can invade and replicate in nonphagocytic host cells, such as alveolar epithelial cells, kidney epithelial cells, hepatocytes, and fibroblasts (14, 21, 25-27, 47). Although there has been recent progress on understanding the bacterial molecules that contribute to intramacrophage growth, less has been done to investigate F. tularensis growth in other cell types. And in the current paradigm of tularemia pathogenesis, F. tularensis mutants deficient in intramacrophage replication should be attenuated for virulence during animal infection.The de novo synthesis of pyrimidines is a central metabolic pathway, as these molecules are precursors of RNA, DNA, cell membranes, and glycosylation substrates (18, 57). This pathway comprises the activity of six enzymatic reactions that culminate in the decarboxylation of orotidine-5′-phosphate to uridine-5′-monophosphate, a step mediated by the protein product of pyrF (2). Prior reports have shown that F. tularensis genes encoding homologs to enzymes mediating initial steps of pyrimidine biosynthesis, such as carAB and pyrB, are essential for intramacrophage growth (47, 56). Although F. tularensis mutants of these genes did not replicate in primary macrophages in vitro, the in vivo virulence of these strains was not detailed fully (56).The gene encoding the ultimate enzyme of pyrimidine biosynthesis, pyrF, has not been characterized for Francisella. pyrF mutations in other bacteria lead to uracil auxotrophy and resistance to 5-fluoroorotic acid (FOA), which provides a nonantibiotic counterselectable marker useful in applied bacterial genetics (30, 45, 54, 55). A counterselectable marker would be especially beneficial when working with a category A biodefense agent, such as F. tularensis, where the choice of antibiotic selection and the cognate resistance markers are limited. An F. tularensis strain containing a deletion of pyrF may be attenuated for virulence and would also be a potential vaccine candidate.In this report, we characterize pyrF mutant strains of both F. tularensis LVS and the fully virulent F. tularensis strain Schu S4. Using a genetic approach, we show that this gene encodes a functional orotidine-5′-phosphate decarboxylase and that its activity is critical for replication in macrophages in vitro. Although ΔpyrF mutants of both LVS and Schu S4 had similar phenotypes, we present evidence suggesting that F. tularensis Schu S4 possesses more mechanisms than LVS for silencing macrophage activation. Surprisingly, the ΔpyrF mutants were not attenuated in vivo. Furthermore, we show that intramacrophage replication is expendable during F. tularensis infection, provided that this bacterium can still replicate in nonmacrophage cells. These findings delineate the contribution of F. tularensis replication in the host''s nonmacrophage cells in vivo and provide novel insight into the pathogenesis of this bacterium.     

Protection afforded against aerosol challenge by systemic immunisation with inactivated Francisella tularensis live vaccine strain (LVS)

BALB/c mice were immunised with inactivated Francisella tularensis live vaccine strain (LVS) and the level of protection afforded against aerosol challenge with virulent strains of F. tularensis ascertained. Intramuscular (IM) injection of inactivated LVS with an aluminium-hydroxide-based adjuvant-stimulated IgG1-biased LVS-specific antibody responses and afforded no protection against aerosol challenge with subspecies holarctica (strain HN63). Conversely, IM injection of inactivated LVS adjuvanted with preformed immune-stimulating complexes (ISCOMS) admixed with immunostimulatory CpG oligonucleotides afforded robust protection against aerosol-initiated infection with HN63. However, despite a significantly extended time-to-death relative to naïve controls, the majority of mice immunised with the most potent vaccine formulation were not protected against a low-dose aerosol challenge with subspecies tularensis (strain Schu S4). These data indicate that parenterally administered non-living vaccines can be used for effective immunisation against aerosol challenges with subspecies holarctica, although not high virulence strains of F. tularensis.     

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.     

Francisella tularensis Live Vaccine Strain Folate Metabolism and Pseudouridine Synthase Gene Mutants Modulate Macrophage Caspase-1 Activation

Francisella tularensis is a Gram-negative bacterium and the causative agent of the disease tularemia. Escape of F. tularensis from the phagosome into the cytosol of the macrophage triggers the activation of the AIM2 inflammasome through a mechanism that is not well understood. Activation of the AIM2 inflammasome results in autocatalytic cleavage of caspase-1, resulting in the processing and secretion of interleukin-1β (IL-1β) and IL-18, which play a crucial role in innate immune responses to F. tularensis. We have identified the 5-formyltetrahydrofolate cycloligase gene (FTL_0724) as being important for F. tularensis live vaccine strain (LVS) virulence. Infection of mice in vivo with a F. tularensis LVS FTL_0724 mutant resulted in diminished mortality compared to infection of mice with wild-type LVS. The FTL_0724 mutant also induced increased inflammasome-dependent IL-1β and IL-18 secretion and cytotoxicity in macrophages in vitro. In contrast, infection of macrophages with a F. tularensis LVS rluD pseudouridine synthase (FTL_0699) mutant resulted in diminished IL-1β and IL-18 secretion from macrophages in vitro compared to infection of macrophages with wild-type LVS. In addition, the FTL_0699 mutant was not attenuated in vivo. These findings further illustrate that F. tularensis LVS possesses numerous genes that influence its ability to activate the inflammasome, which is a key host strategy to control infection with this pathogen in vivo.     

TolC-Dependent Modulation of Host Cell Death by the Francisella tularensis Live Vaccine Strain

Francisella tularensis is a facultative intracellular, Gram-negative pathogen and the causative agent of tularemia. We previously identified TolC as a virulence factor of the F. tularensis live vaccine strain (LVS) and demonstrated that a ΔtolC mutant exhibits increased cytotoxicity toward host cells and elicits increased proinflammatory responses compared to those of the wild-type (WT) strain. TolC is the outer membrane channel component used by the type I secretion pathway to export toxins and other bacterial virulence factors. Here, we show that the LVS delays activation of the intrinsic apoptotic pathway in a TolC-dependent manner, both during infection of primary macrophages and during organ colonization in mice. The TolC-dependent delay in host cell death is required for F. tularensis to preserve its intracellular replicative niche. We demonstrate that TolC-mediated inhibition of apoptosis is an active process and not due to defects in the structural integrity of the ΔtolC mutant. These findings support a model wherein the immunomodulatory capacity of F. tularensis relies, at least in part, on TolC-secreted effectors. Finally, mice vaccinated with the ΔtolC LVS are protected from lethal challenge and clear challenge doses faster than WT-vaccinated mice, demonstrating that the altered host responses to primary infection with the ΔtolC mutant led to altered adaptive immune responses. Taken together, our data demonstrate that TolC is required for temporal modulation of host cell death during infection by F. tularensis and highlight how shifts in the magnitude and timing of host innate immune responses may lead to dramatic changes in the outcome of infection.     

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.     

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.     

A tolC Mutant of Francisella tularensis Is Hypercytotoxic Compared to the Wild Type and Elicits Increased Proinflammatory Responses from Host Cells

The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen and the causative agent of tularemia. TolC, which is an outer membrane protein involved in drug efflux and type I protein secretion, is required for the virulence of the F. tularensis live vaccine strain (LVS) in mice. Here, we show that an LVS ΔtolC mutant colonizes livers, spleens, and lungs of mice infected intradermally or intranasally, but it is present at lower numbers in these organs than in those infected with the parental LVS. For both routes of infection, colonization by the ΔtolC mutant is most severely affected in the lungs, suggesting that TolC function is particularly important in this organ. The ΔtolC mutant is hypercytotoxic to murine and human macrophages compared to the wild-type LVS, and it elicits the increased secretion of proinflammatory chemokines from human macrophages and endothelial cells. Taken together, these data suggest that TolC function is required for F. tularensis to inhibit host cell death and dampen host immune responses. We propose that, in the absence of TolC, F. tularensis induces excessive host cell death, causing the bacterium to lose its intracellular replicative niche. This results in lower bacterial numbers, which then are cleared by the increased innate immune response of the host.Francisella tularensis is the etiological agent of tularemia. F. tularensis is classified as a category A agent of bioterrorism by the U.S. Centers for Disease Control and Prevention (http://emergency.cdc.gov/agent/agentlist-category.asp) due to its low infectious dose, ease of aerosol dissemination, and capacity to cause high morbidity and mortality (19). There are two clinically relevant subspecies of F. tularensis: subsp. tularensis, which is extremely pathogenic in humans, and subsp. holarctica, which causes a less severe clinical presentation (48). The most severe form of the disease is pneumonic tularemia caused by the inhalation of aerosolized F. tularensis subsp. tularensis (19). The F. tularensis subsp. holarctica-derived live vaccine strain (LVS) was used for many years as the vaccination against tularemia. However, the basis for its attenuation is unknown, and it is no longer in use as a vaccine (46). The LVS is highly virulent in mice, where it causes a disease closely resembling human tularemia (30). These features make the LVS an important model for the study of tularemia. An additional Francisella species, F. novicida, causes disease only in immunocompromised individuals. F. novicida, like the LVS, is highly virulent in mice and widely used as a model of tularemia (20).F. tularensis is a Gram-negative, facultative intracellular pathogen (50). Although factors important for the virulence of F. tularensis are beginning to be identified, the molecular mechanisms behind the extreme pathogenicity of this organism still are largely unknown. In vivo, F. tularensis is a stealth pathogen, evading host cell defenses and dampening host proinflammatory responses. F. tularensis produces an unusual lipopolysaccharide that has low toxicity and does not activate host cells in a TLR4-dependent manner (4, 22). A critical aspect of the pathogenesis of F. tularensis is its ability to escape the phagosome and replicate within the cytosol of a variety of host cells, including both murine and human macrophages and dendritic cells (2, 3, 16, 25, 49). Although F. tularensis does have an extracellular phase (24), it is thought that cytosolic replication allows the bacteria to grow to large numbers while avoiding detection by the host immune system.Host cells respond to F. tularensis invasion by inducing cell death pathways, including apoptosis and pyroptosis (32, 38). In the intrinsic apoptotic pathway, cytochrome c is released from mitochondria into the cytosol, leading to caspase-9 activation and ultimately to the activation of effector caspases such as caspase-3 and -7 (10). In pyroptosis, caspase-1 is activated through the inflammasome complex, resulting in the release of proinflammatory cytokines such as interleukin-1ß (IL-1ß) (6, 32). Lai and coworkers demonstrated that the infection of murine J774 macrophage-like cells with the LVS activated the intrinsic apoptotic pathway as early as 12 h postinfection. Activated caspase-3, but not caspase-1, was detected in the infected cells (38). In contrast, Mariathasan et al. found that the infection of preactivated murine peritoneal macrophages by either the LVS or strain U112 (F. novicida) triggered pyroptosis and the release of IL-1ß (42). In both studies, the induction of cell death was dependent upon the bacteria escaping the phagosome and initiating cytosolic replication. Weiss and colleagues isolated mutants of strain U112 that were attenuated in vivo and caused increased cell death in tissue culture compared to that caused by wild-type U112 (53). This suggests that although host cells initiate death pathways in response to F. tularensis infection, the bacteria has the ability to actively reduce cell death, and this is important for virulence.In addition to triggering death pathways, host cells respond to invading bacteria by mounting a proinflammatory response to alert neighboring cells of the impending bacterial threat (17). However, F. tularensis has been shown to actively suppress these innate host responses. Telepnev and coworkers showed that the LVS disrupted toll-like receptor signaling and blocked the secretion of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1ß by murine and human macrophages (51, 52). Similarly, Bosio and colleagues showed that the LVS inhibited the innate immune response of murine pulmonary dendritic cells to bacterial ligands, and the infection of mice with the fully virulent Schu4 strain (F. tularensis subsp. tularensis) caused an overall state of immunosuppression in the lungs (8, 9).The genome analysis of F. tularensis identified only a few potential virulence factors, suggesting that the bacterium uses novel factors to achieve its high level of pathogenicity (40). Unique to F. tularensis is a 33.9-kb region of DNA termed the Francisella pathogenicity island (FPI) (29, 40, 45). The FPI encodes genes that are essential for intracellular survival and virulence, including iglABCD and pdpABCD (45). F. tularensis lacks type III and IV secretion systems, which is surprising considering its intracellular nature. These secretion systems commonly are used by intracellular pathogens to deliver effector proteins inside host cells to manipulate host cell responses (14, 26). F. tularensis does contain genes encoding a type IV pilus biogenesis system that also functions in the secretion of soluble proteins by a type II-like mechanism and that are important for virulence (12, 31, 54). Finally, F. tularensis appears to contain a functioning type I secretion system that is critical for pathogenesis (28).Type I secretion systems function in the secretion of a variety of toxins and other virulence factors directly from the cytoplasm to the extracellular milieu in a single energized step (33, 37). The type I system consists of three separate components: an outer membrane channel-forming protein, a periplasmic adaptor or membrane fusion protein, and an inner membrane pump that typically belongs to the ATP-binding cassette family. The TolC protein of Escherichia coli, which functions in hemolysin secretion, is the prototypical outer membrane channel component (37). In addition to protein secretion, TolC functions in the efflux of small noxious molecules, conferring multidrug resistance (37). F. tularensis contains three TolC paralogs, TolC, FtlC, and SilC, with TolC and FtlC exhibiting significant homology to the E. coli TolC protein (28, 35). In a previous study we created tolC and ftlC deletion mutants in the F. tularensis LVS (28). We found that both TolC and FtlC participate in multidrug resistance in F. tularensis, but only the ΔtolC mutant was attenuated for virulence in mice by the intradermal route. Thus, tolC is a critical virulence factor of F. tularensis and likely functions in type I secretion in addition to multidrug efflux.Here, we delineate the molecular mechanisms behind the attenuation of the LVS ΔtolC mutant in mice infected by both the intradermal and intranasal routes. In vivo organ burden assays revealed that the ΔtolC strain is decreased for the bacterial colonization of liver, spleen, and most prominently, lungs. In vitro experiments revealed that the ΔtolC mutant is hypercytotoxic to murine macrophages, causing increased apoptosis via a mechanism involving caspase-3 but not caspase-1. In addition, the LVS ΔtolC mutant was hypercytotoxic toward human macrophages and elicited the significantly increased secretion of the proinflammatory chemokines CXCL8 (also known as IL-8) and CCL2 (also known as monocyte chemoattractant protein [MCP-1]). Taken together, these data demonstrate a critical role for TolC, likely via a TolC-secreted toxin(s), in the successful intracellular lifestyle of F. tularensis, its ability to evade host innate immune responses, and its overall virulence.     

Infection with Francisella tularensis LVS clpB Leads to an Altered yet Protective Immune Response

Bacterial attenuation is typically thought of as reduced bacterial growth in the presence of constant immune pressure. Infection with Francisella tularensis elicits innate and adaptive immune responses. Several in vivo screens have identified F. tularensis genes necessary for virulence. Many of these mutations render F. tularensis defective for intracellular growth. However, some mutations have no impact on intracellular growth, leading us to hypothesize that these F. tularensis mutants are attenuated because they induce an altered host immune response. We were particularly interested in the F. tularensis LVS (live vaccine strain) clpB (FTL_0094) mutant because this strain was attenuated in pneumonic tularemia yet induced a protective immune response. The attenuation of LVS clpB was not due to an intracellular growth defect, as LVS clpB grew similarly to LVS in primary bone marrow-derived macrophages and a variety of cell lines. We therefore determined whether LVS clpB induced an altered immune response compared to that induced by LVS in vivo. We found that LVS clpB induced proinflammatory cytokine production in the lung early after infection, a process not observed during LVS infection. LVS clpB provoked a robust adaptive immune response similar in magnitude to that provoked by LVS but with increased gamma interferon (IFN-γ) and interleukin-17A (IL-17A) production, as measured by mean fluorescence intensity. Altogether, our results indicate that LVS clpB is attenuated due to altered host immunity and not an intrinsic growth defect. These results also indicate that disruption of a nonessential gene(s) that is involved in bacterial immune evasion, like F. tularensis clpB, can serve as a model for the rational design of attenuated vaccines.     

Mast cell/IL-4 control of Francisella tularensis replication and host cell death is associated with increased ATP production and phagosomal acidification

Mast cells are now recognized as effective modulators of innate immunity. We recently reported that mast cells and secreted interleukin-4 (IL-4) effectively control intramacrophage replication of Francisella tularensis Live Vaccine Strain (LVS), and that mice deficient in mast cells or IL-4 receptor (IL-4R^−/−) exhibit greater susceptibility to pulmonary challenge. In this study, we further evaluated the mechanism(s) by which mast cells/IL-4 control intramacrophage bacterial replication and host cell death, and found that IL-4R^−/− mice exhibited significantly greater induction of active caspase-3 within lung macrophages than wild-type animals following intranasal challenge with either LVS or the human virulent type A strain SCHU S4. Treatment of LVS-infected bone-marrow-derived macrophages with a pancaspase inhibitor (zVAD) did not alter bacterial replication, but minimized active caspase-3 and other markers (Annexin V and propidium iodide) of cell death, whereas treatment with both rIL-4 and zVAD resulted in concomitant reduction of both parameters, suggesting that inhibition of bacterial replication by IL-4 was independent of caspase activation. Interestingly, IL-4-treated infected macrophages exhibited significantly increased ATP production and phagolysosomal acidification, as well as enhanced mannose receptor upregulation and increased internalization with acidification, which correlated with observations in mast cell-macrophage co-cultures, with resultant decreases in F. tularensis replication.     

A Francisella tularensis Live Vaccine Strain (LVS) Mutant with a Deletion in capB,Encoding a Putative Capsular Biosynthesis Protein,Is Significantly More Attenuated than LVS yet Induces Potent Protective Immunity in Mice against F. tularensis Challenge

Francisella tularensis, the causative agent of tularemia, is in the top category (category A) of potential agents of bioterrorism. The F. tularensis live vaccine strain (LVS) is the only vaccine currently available to protect against tularemia; however, this unlicensed vaccine is relatively toxic and provides incomplete protection against aerosolized F. tularensis, the most dangerous mode of transmission. Hence, a safer and more potent vaccine is needed. As a first step toward addressing this need, we have constructed and characterized an attenuated version of LVS, LVS ΔcapB, both as a safer vaccine and as a vector for the expression of recombinant F. tularensis proteins. LVS ΔcapB, with a targeted deletion in a putative capsule synthesis gene (capB), is antibiotic resistance marker free. LVS ΔcapB retains the immunoprotective O antigen, is serum resistant, and is outgrown by parental LVS in human macrophage-like THP-1 cells in a competition assay. LVS ΔcapB is significantly attenuated in mice; the 50% lethal dose (LD₅₀) intranasally (i.n.) is >10,000-fold that of LVS. Providing CapB in trans to LVS ΔcapB partially restores its virulence in mice. Mice immunized with LVS ΔcapB i.n. or intradermally (i.d.) developed humoral and cellular immune responses comparable to those of mice immunized with LVS, and when challenged 4 or 8 weeks later with a lethal dose of LVS i.n., they were 100% protected from illness and death and had significantly lower levels (3 to 5 logs) of LVS in the lung, liver, and spleen than sham-immunized mice. Most importantly, mice immunized with LVS ΔcapB i.n. or i.d. and then challenged 6 weeks later by aerosol with 10× the LD₅₀ of the highly virulent type A F. tularensis strain SchuS4 were significantly protected (100% survival after i.n. immunization). These results show that LVS ΔcapB is significantly safer than LVS and yet provides potent protective immunity against virulent F. tularensis SchuS4 challenge.Francisella tularensis is a Gram-negative coccobacillus that causes tularemia, a zoonotic disease spread among small animals such as rabbits and rodents by blood-sucking insects. Humans typically acquire tularemia by handling infected animals or from the bite of infected insects. There are four subspecies of F. tularensis: F. tularensis subsp. tularensis, holarctica, mediasiatica, and novicida (41); of these, F. tularensis subsp. tularensis, found in North America and also known as type A, causes the most severe disease. Following cutaneous exposure, tularemia typically presents as an ulceronodular disease with painful, ulcerated skin lesions and swollen lymph nodes. Following inhalation exposure, tularemia presents with acute flu-like symptoms followed by pleuropneumonic and typhoidal illness. The pneumonic form of tularemia has a high fatality rate (11).Because of its high pathogenicity in humans, especially after respiratory exposure, its low infectious dose, and the relative ease with which it can be cultured and disseminated, F. tularensis is classified as a category A agent of bioterrorism, i.e., among bioterrorist agents thought to pose the greatest risk to the public. Indeed, F. tularensis was previously developed as a bioweapon and stockpiled by Japan during World War II (16) and by the United States and the Soviet Union during the Cold War (1, 6). Although tularemia can be treated with available antibiotics, F. tularensis can be genetically engineered to be antibiotic resistant (30). Moreover, pneumonic tularemia frequently requires hospitalization and intensive care, and even when an infected individual is treated with antibiotics to which the organism is sensitive, the disease may resolve slowly (12); even a moderately sized outbreak could rapidly overwhelm medical facilities (11). Hence, relying on antibiotics to protect against a bioterrorist attack with F. tularensis is not a practical public health approach. A safe and potent vaccine, on the other hand, would appear to offer a much more reliable approach.An unlicensed vaccine known as the live vaccine strain (LVS), an attenuated mutant of F. tularensis subsp. holarctica, was developed in the mid-1900s and is the only vaccine currently available in the United States. The underlying mechanism of attenuation is not fully characterized genetically, although recently, the reintroduction of deleted genes pilA and FTT0918 was shown to restore virulence to the level of virulent type B strains (35). The LVS vaccine has several drawbacks. The vaccine, which retains considerable virulence in animals, shows significant toxicity in humans after both intradermal (i.d.) and aerosol administration (19, 37). Moreover, it provides incomplete protection to humans challenged with type A F. tularensis by aerosol, the route of transmission of greatest concern in a bioterrorist attack (19, 29, 37).In a search for a vaccine that is safer and more potent than LVS, we sought to rationally attenuate LVS and to use the attenuated LVS as both a vaccine and a vector to overexpress immunogenic F. tularensis proteins. We hypothesized that we would render LVS safer by further attenuating it and that we would render it more potent by overexpressing key immunoprotective antigens. This overall strategy mirrors that used successfully to develop the first vaccine against tuberculosis that is more potent than the current Mycobacterium bovis BCG vaccine, rBCG30, a recombinant BCG vaccine overexpressing the Mycobacterium tuberculosis 30-kDa major secretory protein, and to develop the first vaccine both safer and more potent than BCG, rBCG(mbtB)30, an attenuated version of rBCG30 that is engineered and propagated such that it can multiply only a few times in the host (20, 21, 45).In attenuating LVS, we sought a mutation that would greatly reduce virulence but have a minimal impact on immunogenicity and protective efficacy. Transposon mutagenesis studies of F. tularensis subsp. novicida and holarctica (LVS) have shown that mutants with transposon insertions in genes (FTT0806, FTT0805, and FTT0798) encoding proteins putatively involved in capsular biosynthesis, on the basis of partial amino acid sequence homology with capsular biosynthesis proteins of Bacillus anthracis, are highly attenuated (∼100- to 1,000-fold) in mice (43, 47). Consequently, we decided to evaluate the vaccine potential of an LVS mutant with a deletion in one of these genes.In this study, we describe the construction of an antibiotic resistance marker-free FTL_1416/FTT0805 (capB) deletion mutant of F. tularensis LVS (LVS ΔcapB) and show that LVS ΔcapB is resistant to serum killing, outgrown by its parental LVS in human macrophage-like THP-1 cells, and highly attenuated in mice. We demonstrate further that this vaccine, after both i.d. and intranasal (i.n.) administration, induces potent cellular and humoral immune responses and significant protective immunity against respiratory challenge with virulent F. tularensis.