Construction and characterization of an attenuated purine auxotroph in a Francisella tularensis live vaccine strain

Francisella tularensis is a facultative intracellular pathogen and is the etiological agent of tularemia. It is capable of escaping from the phagosome, replicating to high numbers in the cytosol, and inducing apoptosis in macrophages of a variety of hosts. F. tularensis has received significant attention recently due to its potential use as a bioweapon. Currently, there is no licensed vaccine against F. tularensis, although a partially protective live vaccine strain (LVS) that is attenuated in humans but remains fully virulent for mice was previously developed. An F. tularensis LVS mutant deleted in the purMCD purine biosynthetic locus was constructed and partially characterized by using an allelic exchange strategy. The F. tularensis LVS delta purMCD mutant was auxotrophic for purines when grown in defined medium and exhibited significant attenuation in virulence when assayed in murine macrophages in vitro or in BALB/c mice. Growth and virulence defects were complemented by the addition of the purine precursor hypoxanthine or by introduction of purMCDN in trans. The F. tularensis LVS delta purMCD mutant escaped from the phagosome but failed to replicate in the cytosol or induce apoptotic and cytopathic responses in infected cells. Importantly, mice vaccinated with a low dose of the F. tularensis LVS delta purMCD mutant were fully protected against subsequent lethal challenge with the LVS parental strain. Collectively, these results suggest that F. tularensis mutants deleted in the purMCD biosynthetic locus exhibit characteristics that may warrant further investigation of their use as potential live vaccine candidates.     

Role of the wbt locus of Francisella tularensis in lipopolysaccharide O-antigen biogenesis and pathogenicity

Francisella tularensis is a highly infectious bacterial pathogen, responsible for the zoonotic disease tularemia. We screened a bank of transposon insertion mutants of F. tularensis subsp. holarctica LVS for colony morphology alterations and selected a mutant with a transposon insertion in wbtA, the first gene of the predicted lipopolysaccharide O-antigen gene cluster. Inactivation of wbtA led to the complete loss of O antigen, conferred serum sensitivity, impaired intracellular replication, and severely attenuated virulence in the mouse model. Notably, this mutant afforded protection against a challenge against virulent LVS.     

Members of the Francisella tularensis phagosomal transporter subfamily of major facilitator superfamily transporters are critical for pathogenesis

Francisella tularensis is the causative agent of tularemia. Due to its aerosolizable nature and low infectious dose, F. tularensis is classified as a category A select agent and, therefore, is a priority for vaccine development. Survival and replication in macrophages and other cell types are critical to F. tularensis pathogenesis, and impaired intracellular survival has been linked to a reduction in virulence. The F. tularensis genome is predicted to encode 31 major facilitator superfamily (MFS) transporters, and the nine-member Francisella phagosomal transporter (Fpt) subfamily possesses homology with virulence factors in other intracellular pathogens. We hypothesized that these MFS transporters may play an important role in F. tularensis pathogenesis and serve as good targets for attenuation and vaccine development. Here we show altered intracellular replication kinetics and attenuation of virulence in mice infected with three of the nine Fpt mutant strains compared with wild-type (WT) F. tularensis LVS. The vaccination of mice with these mutant strains was protective against a lethal intraperitoneal challenge. Additionally, we observed pronounced differences in cytokine profiles in the livers of mutant-infected mice, suggesting that alterations in in vivo cytokine responses are a major contributor to the attenuation observed for these mutant strains. These results confirm that this subset of MFS transporters plays an important role in the pathogenesis of F. tularensis and suggest that a focus on the development of attenuated Fpt subfamily MFS transporter mutants is a viable strategy toward the development of an efficacious vaccine.     

A conserved and immunodominant lipoprotein of Francisella tularensis is proinflammatory but not essential for virulence

Francisella tularensis is a highly virulent bacterium that causes tularemia, a disease that is often fatal if untreated. A live vaccine strain (LVS) of this bacterium is attenuated for virulence in humans but produces lethal disease in mice. F. tularensis has been classified as a Category A agent of bioterrorism. Despite this categorization, little is known about the components of the organism that are responsible for causing disease in its hosts. Here, we report the deletion of a well-characterized lipoprotein of F. tularensis, designated LpnA (also known as Tul4), in the LVS. An LpnA deletion mutant was comparable to the wild-type strain in its ability to grow intracellularly and cause lethal disease in mice. Additionally, mice inoculated with a sublethal dose of the mutant strain were afforded the same protection against a subsequent lethal challenge with the LVS as were mice initially administered a sublethal dose of the wild-type bacterium. The LpnA-deficient strain showed an equivalent ability to promote secretion of chemokines by human monocyte-derived macrophages as its wild-type counterpart. However, recombinant LpnA potently stimulated primary cultures of human macrophages in a Toll-like receptor 2-dependent manner. Although human endothelial cells were also activated by recombinant LpnA, their response was relatively modest. LpnA is clearly unnecessary for multiple functions of the LVS, but its inflammatory capacity implicates it and other Francisella lipoproteins as potentially important to the pathogenesis of tularemia.     

A Francisella tularensis locus required for spermine responsiveness is necessary for virulence

Tularemia is a debilitating febrile illness caused by the category A biodefense agent Francisella tularensis. This pathogen infects over 250 different hosts, has a low infectious dose, and causes high morbidity and mortality. Our understanding of the mechanisms by which F. tularensis senses and adapts to host environments is incomplete. Polyamines, including spermine, regulate the interactions of F. tularensis with host cells. However, it is not known whether responsiveness to polyamines is necessary for the virulence of the organism. Through transposon mutagenesis of F. tularensis subsp. holarctica live vaccine strain (LVS), we identified FTL_0883 as a gene important for spermine responsiveness. In-frame deletion mutants of FTL_0883 and FTT_0615c, the homologue of FTL_0883 in F. tularensis subsp. tularensis Schu S4 (Schu S4), elicited higher levels of cytokines from human and murine macrophages compared to wild-type strains. Although deletion of FTL_0883 attenuated LVS replication within macrophages in vitro, the Schu S4 mutant with a deletion in FTT_0615c replicated similarly to wild-type Schu S4. Nevertheless, both the LVS and the Schu S4 mutants were significantly attenuated in vivo. Growth and dissemination of the Schu S4 mutant was severely reduced in the murine model of pneumonic tularemia. This attenuation depended on host responses to elevated levels of proinflammatory cytokines. These data associate responsiveness to polyamines with tularemia pathogenesis and define FTL_0883/FTT_0615c as an F. tularensis gene important for virulence and evasion of the host immune response.     

Modulation of virulence factors in Francisella tularensis determines human macrophage responses

Francisella tularensis, the causative agent of tularemia and Category A biodefense agent, is known to replicate within host macrophages, though the pathogenesis of this organism is incompletely understood. We have isolated a variant of F. tularensis live vaccine strain (LVS) based on colony morphology and its effect on macrophages. Human monocyte-derived macrophages produced more tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1beta, IL-6, and IL-12 p40 following exposure to the variant, designated the activating variant (ACV). The immunoreactivity of the lipopolysaccharide (LPS) from both LVS and ACV was comparable to the previously described blue variant and was distinct from the gray variant of LVS. We found, however, the soluble protein fractions of LVS and ACV differed. Further investigation using two-dimensional gel electrophoresis demonstrated higher levels of several proteins in the parental LVS isolate. The differentially expressed proteins featured several associated with virulence in F. tularensis and other pathogens, including intracellular growth locus C (IglC), a sigma(54)-modulation protein family member (YhbH), and aconitase. ACV reverted to the LVS phenotype, indicated by low cytokine induction and high IglC expression, after growth in a chemically defined medium. These data provide evidence that the levels of virulence factors in F. tularensis are modulated based on culture conditions and that this modulation impacts host responses. This work provides a basis for investigation of Francisella virulence factor regulation and the identification of additional factors, co-regulated with IglC, that affect macrophage responses.     

Francisella tularensis LVS evades killing by human neutrophils via inhibition of the respiratory burst and phagosome escape

Francisella tularensis is a Gram-negative bacterium and the causative agent of tularemia. Recent data indicate that F. tularensis replicates inside macrophages, but its fate in other cell types, including human neutrophils, is unclear. We now show that F. tularensis live vaccine strain (LVS), opsonized with normal human serum, was rapidly ingested by neutrophils but was not eliminated. Moreover, evasion of intracellular killing can be explained, in part, by disruption of the respiratory burst. As judged by luminol-enhanced chemiluminescence and nitroblue tetrazolium staining, neutrophils infected with live F. tularensis did not generate reactive oxygen species. Confocal microscopy demonstrated that NADPH oxidase assembly was disrupted, and LVS phagosomes did not acquire gp91/p22(phox) or p47/p67(phox). At the same time, F. tularensis also impaired neutrophil activation by heterologous stimuli such as phorbol esters and opsonized zymosan particles. Later in infection, LVS escaped the phagosome, and live organisms persisted in the neutrophil cytosol for at least 12 h. To our knowledge, our data are the first demonstration of a facultative intracellular pathogen, which disrupts the oxidative burst and escapes the phagosome to evade elimination inside neutrophils, and as such, our data define a novel mechanism of virulence.     

Virulence of Francisella spp. in chicken embryos

We examined the utility of infecting chicken embryos as a means of evaluating the virulence of different Francisella sp. strains and mutants. Infection of 7-day-old chicken embryos with a low dose of F. novicida or F. tularensis subsp. holarctica live vaccine strain (LVS) resulted in sustained growth for 6 days. Different doses of these two organisms were used to inoculate chicken embryos to determine the time to death. These experiments showed that wild-type F. novicida was at least 10,000-fold more virulent than the LVS strain. We also examined the virulence of several attenuated mutants of F. novicida, and they were found to have a wide range of virulence in chicken embryos. Fluorescent microscopic examination of infected chicken embryo organs revealed that F. tularensis grew in scattered foci of infections, and in all cases the F. tularensis appeared to be growing intracellularly. These results demonstrate that infection of 7-day-old chicken embryos can be used to evaluate the virulence of attenuated F. tularensis strains.     

Identification of Francisella tularensis genes affected by iron limitation

Cells of an attenuated live vaccine strain (LVS) of F. tularensis grown under iron-restricted conditions were found to contain increased quantities of several proteins relative to cells of this same strain grown under iron-replete conditions. Mass spectrometric analysis identified two of these proteins as IglC and PdpB, both of which are encoded by genes located in a previously identified pathogenicity island in F. tularensis LVS. Regions with homology to the consensus Fur box sequence were located immediately in front of the iglC and pdpB open reading frames (ORFs), and in silico analysis of the F. tularensis Schu4 genome detected a number of predicted 5' untranslated regions that contained putative Fur boxes. The putative Fur box preceding Francisella iron-regulated gene A (figA) had the highest degree of identity with the consensus Fur box sequence. DNA microarray analysis showed that nearly 80 of the genes in the F. tularensis LVS genome were up- or down-regulated at least twofold under iron-restricted growth conditions. When tested for possible siderophore production by means of the Chrome Azurol S assay, a wild-type F. novicida strain produced a large reaction zone whereas its figA mutant produced very little reactivity in this assay. In addition, a cross-feeding experiment demonstrated that this siderophore-like activity produced by the wild-type F. novicida strain could enhance the ability of the F. novicida figA mutant to grow under iron-restricted conditions. This study provides the first identification of iron-regulated genes in F. tularensis LVS and evidence for the production of a siderophore-like molecule by F. novicida.     

Potential source of Francisella tularensis live vaccine strain attenuation determined by genome comparison

Francisella tularensis is a bacterial pathogen that causes the zoonotic disease tularemia and is important to biodefense. Currently, the only vaccine known to confer protection against tularemia is a specific live vaccine strain (designated LVS) derived from a virulent isolate of Francisella tularensis subsp. holarctica. The origin and source of attenuation of this strain are not known. To assist with the design of a defined live vaccine strain, we sought to determine the genetic basis of the attenuation of LVS. This analysis relied primarily on the comparison between the genome of LVS and Francisella tularensis holarctica strain FSC200, which differ by only 0.08% of their nucleotide sequences. Under the assumption that the attenuation was due to a loss of function(s), only coding regions were examined in this comparison. To complement this analysis, the coding regions of two slightly more distantly related Francisella tularensis strains were also compared against the LVS coding regions. Thirty-five genes show unique sequence variations predicted to alter the protein sequence in LVS compared to the other Francisella tularensis strains. Due to these polymorphisms, the functions of 15 of these genes are very likely lost or impaired. Seven of these genes were demonstrated to be under stronger selective constraints, suggesting that they are the most probable to be the source of LVS attenuation and useful for a newly defined vaccine.     

Type IV pili in Francisella tularensis: roles of pilF and pilT in fiber assembly, host cell adherence, and virulence

Francisella tularensis, a highly virulent facultative intracellular bacterium, is the causative agent of tularemia. Genome sequencing of all F. tularensis subspecies revealed the presence of genes that could encode type IV pili (Tfp). The live vaccine strain (LVS) expresses surface fibers resembling Tfp, but it was not established whether these fibers were indeed Tfp encoded by the pil genes. We show here that deletion of the pilF putative Tfp assembly ATPase in the LVS resulted in a complete loss of surface fibers. Disruption of the pilT putative disassembly ATPase also caused a complete loss of pili, indicating that pilT functions differently in F. tularensis than in model Tfp systems such as those found in Pseudomonas aeruginosa and Neisseria spp. The LVS pilF and pilT mutants were attenuated for virulence in a mouse model of tularemia by the intradermal route. Furthermore, although absence of pili had no effect on the ability of the LVS to replicate intracellularly, the pilF and pilT mutants were defective for adherence to macrophages, pneumocytes, and hepatocytes. This work confirms that the surface fibers expressed by the LVS are encoded by the pil genes and provides evidence that the Francisella pili contribute to host cell adhesion and virulence.     

Identification of immunoreactive antigens in membrane proteins enriched fraction from Francisella tularensis LVS

Francisella tularensis is a Gram-negative, facultative intracellular bacterium causing disease in many mammalian species. The low infectious dose of F. tularensis and the ease of air-borne transmission are the main features responsible for the classification of this bacterium as a potential biological weapon. The live attenuated strain of F. tularensis live vaccine strain (LVS) is currently only effective vaccine against tularemia, however, this type of vaccine has not been approved for human use. In the presented study, sub-immunoproteome analysis was performed to search for new immunogenic proteins of Francisella tularensis LVS grown under different conditions. By this approach 35 immunoreactive antigens were identified, 19 of them showed to be novel immunogens. In conclusion, sub-immunoproteome analysis resulted in successful identification of novel immunoreactive proteins.     

Francisella tularensis enters macrophages via a novel process involving pseudopod loops

Intracellular bacterial pathogens employ a variety of strategies to invade their eukaryotic host cells. From an ultrastructural standpoint, the processes that bacteria employ to invade their host cells include conventional phagocytosis, coiling phagocytosis, and ruffling/triggered macropinocytosis. In this paper, we describe a novel process by which Francisella tularensis, the agent of tularemia, enters host macrophages. F. tularensis is a remarkably infectious facultative intracellular bacterial parasite--as few as 10 bacteria can cause life-threatening disease in humans. However, the ultrastructure of its uptake and the receptor mechanisms that mediate its uptake have not been reported previously. We have used fluorescence microscopy and electron microscopy to examine the adherence and uptake of a virulent recent clinical isolate of F. tularensis, subspecies tularensis, and the live vaccine strain (LVS), subspecies holarctica, by human macrophages. We show here that both strains of F. tularensis enter human macrophages by a novel process of engulfment within asymmetric, spacious pseudopod loops, a process that differs ultrastructurally from all previously described uptake mechanisms. We demonstrate also that adherence and uptake of F. tularensis by macrophages is strongly dependent upon complement receptors and upon serum with intact complement factor C3 and that uptake requires actin microfilaments. These findings have significant implications for understanding the intracellular biology and virulence of this extremely infectious pathogen.     

Inactivated Francisella tularensis live vaccine strain protects against respiratory tularemia by intranasal vaccination in an immunoglobulin A-dependent fashion

Francisella tularensis is a gram-negative intracellular bacterium that is considered to be a potential category A biological weapon due to its extreme virulence. Although vaccination with the attenuated live vaccine strain (LVS) of F. tularensis can protect against lethal challenge, use of inactivated or subunit forms as vaccine candidates for induction of protective antibody responses has not been fully evaluated. In the present study, we examined whether immune protection in the lung could be stimulated by intranasal administration of inactivated LVS together with interleukin-12 (IL-12) as an adjuvant. LVS was inactivated by heat, paraformaldehyde treatment, or exposure to UV, and inactivation of the preparations was confirmed by assessing bacterial growth and the survival of mice after direct inoculation. We found that mucosal vaccination with inactivated LVS provided 90 to 100% protection in mice after lethal intranasal challenge with 10(4) CFU of LVS, and this protection was dependent on inclusion of exogenous IL-12 during vaccine administration. Survival of vaccinated mice after live bacterial challenge was correlated with reduced bacterial burden, decreased pulmonary inflammation, increased serum antibody titers, and lower levels of gamma interferon (IFN-gamma), tumor necrosis factor alpha, and IL-6 in the lungs, livers, and spleens. Whereas NK cells were primarily responsible for the production of IFN-gamma in unvaccinated, challenged animals, vaccinated mice had increased levels of lung IFN-gamma+ CD4+ T cells after challenge. Significantly, mice genetically deficient in immunoglobulin A (IgA) expression were unable to survive lethal challenge after vaccination. These results are the first results to demonstrate that IgA-mediated protection against lethal respiratory tularemia occurs after mucosal vaccination with inactivated F. tularensis LVS.     

A capsule-deficient mutant of Francisella tularensis LVS exhibits enhanced sensitivity to killing by serum but diminished sensitivity to killing by polymorphonuclear leukocytes

The live vaccine strain (LVS) of Francisella tularensis is killed by human polymorphonuclear leukocytes as a result of strictly oxygen-dependent mechanisms (S. L?fgren, A. T?rnvik, M. Thore, and J. Carlsson, Infect. Immun. 43:730-734, 1984). We now report that a capsule-deficient (Cap-) mutant of LVS survives in the leukocytes. In contrast to the encapsulated parent strain, the Cap- mutant was avirulent in mice and was susceptible to the bactericidal effect of nonimmune human serum. The mutant was killed by serum as a result of activation of the classical pathway of complement by naturally occurring immunoglobulin M. This killing by serum was mitigated by the presence of human polymorphonuclear leukocytes. After opsonization in complement component C5-deficient nonimmune serum, the Cap- mutant was ingested and survived in the leukocytes. Under these conditions, the parent strain was killed. The leukocytes responded to both the parent and the Cap- strain with a very low chemiluminescent response. Only the response to the parent strain was inhibited by superoxide dismutase. When the Cap- mutant was opsonized with immunoglobulin G, it induced a higher and superoxide dismutase-inhibitable chemiluminescent response and was killed by the leukocytes. In conclusion, the capsule of F. tularensis LVS seemed to protect this organism against the bactericidal effect of serum. When deprived of the capsule, the organism failed to induce an antimicrobial response in polymorphonuclear leukocytes and survived in the leukocytes. Survival in phagocytes is a key characteristic of intracellular parasites. The Cap- mutant of F. tularensis may become a useful tool in experiments to explain the differences between pathways of ingestion of intracellular parasites, evidenced by the death or survival of the parasite.     

Expression of IglC is necessary for intracellular growth and induction of apoptosis in murine macrophages by Francisella tularensis

Francisella tularensis is a facultative intracellular bacterium capable of inducing apoptosis in murine macrophages. In a previous study, an iglC null mutant of F. tularensis live vaccine strain LVS was generated by allelic replacement and in the current study this iglC mutant was successfully complemented in trans. We characterized the capacity of this iglC mutant and the complemented strain to induce macrophage apoptosis. The iglC mutant did not induce apoptosis in the infected cells. In contrast, the complemented iglC strain was able to multiply in the murine macrophage-like cell line J774A.1 and induced apoptosis similar to that of the wild-type strain. It is the first successful example of complementation in trans of a F. tularensis mutant strain and more importantly this work provides direct evidence that the intracellular growth ability is essential for F. tularensis to induce macrophage apoptosis.     

Natural killer and CD8 T cells dominate the response by human peripheral blood mononuclear cells to inactivated Francisella tularensis live vaccine strain

Francisella tularensis is a category A biothreat agent, and as a result, it has recently generated much research interest. F. tularensis live vaccine strain (LVS) is an attenuated form of the virulent F. tularensis organism and has previously been used as a vaccine. However, because of safety concerns, it is no longer approved for this purpose. Thus, the use of inactivated organisms is preferable for vaccine purposes. Although many studies have been performed that examine human peripheral blood mononuclear cells (PBMC), and in particular CD4 T cells, responses to inactivated F. tularensis, there has been no study identifying the individual human cell populations within a mixed PBMC population that respond to this organism. We sought to address this deficit. Our results indicate that natural killer and CD8 T cells comprise the majority of cells responding to F. tularensis LVS. In addition, data suggest CD8 T cell responses are maximal when antibiotic-treated organisms are used and are minimal when formaldehyde-fixed organisms are used. Given the belief that CD8 T cells can play an important role in protection against F. tularensis infection, these studies have direct relevance to the development of F. tularensis vaccines that use inactivated organisms. In addition, important new knowledge is added to our understanding of the human immune response to F. tularensis LVS.     

Francisella tularensis LVS initially activates but subsequently down-regulates intracellular signaling and cytokine secretion in mouse monocytic and human peripheral blood mononuclear cells

Monocytic cells constitute an important defense mechanism against invading pathogens by recognizing conserved pathogens components. The recognition leads to activation of intracellular pathways involving nuclear factor kappa B (NF-kappaB) and mitogen-activated protein kinases (MAPK), such as the c-Jun NH2-terminal kinase (JNK), and p38. We show that in vitro infection with Francisella tularensis results in activation of NF-kappaB, phosphorylation of p38 and c-Jun, and secretion of TNF-alpha in adherent mouse peritoneal cells, in the mouse macrophage-like cell line J774A.1, in the human macrophage cell line THP-1, and in human peripheral blood monocytic cells. This occurred after infection with the human live vaccine strain, F. tularensis LVS or a mutant strain denoted deltaiglC, which lacks expression of a 23-kDa protein, or after addition of killed F. tularensis LVS. Addition of purified F. tularensis LPS resulted in no discernible effects on the cells. When the effects were followed up to 5 h, activation persisted in cultures with killed bacteria or infected with the deltaiglC strain. In contrast, the signal transduction activation and secretion of TNF-alpha were down-regulated within the 5h period in mouse peritoneal cells, J774 cells or human peripheral blood mononuclear cells infected with F. tularensis LVS. Together, the results suggest that infection with live F. tularensis LVS bacteria leads to a rapid induction of a proinflammatory response in mouse and human cells but after internalization of bacteria, this response is completely or partly down-regulated in most cell types. This down-regulation does not occur when cells are infected with the mutant deltaiglC.