Mice sublethally infected with Francisella novicida U112 develop only marginal protective immunity against systemic or aerosol challenge with virulent type A or B strains of F. tularensis

The current study determined the ability of Francisella novicida to act as a live vaccine against the much more virulent, but closely related pathogen, Francisella tularensis. Live attenuated strains of the latter are effective vaccines against human tularemia. However, the molecular cause of their attenuation remains unknown, and this is a regulatory barrier for licensing such vaccines. Moreover, F. tularensis is exceptionally difficult to manipulate genetically. This is hampering the development of rationally attenuated vaccine strains. F. novicida shares a lot of genetic homology with F. tularensis and is more amenable to genetic manipulation. If the former naturally expresses the protective antigens of the latter, it could be used to develop a defined tularemia vaccine. However, the results presented herein show that wild-type F. novicida elicits almost no protection in mice against challenge with virulent F. tularensis.     

Perforin- and granzyme-mediated cytotoxic effector functions are essential for protection against Francisella tularensis following vaccination by the defined F. tularensis subsp. novicida ΔfopC vaccine strain

A licensed vaccine against Francisella tularensis is currently not available. Two Francisella tularensis subsp. novicida (herein referred to by its earlier name, Francisella novicida) attenuated strains, the ΔiglB and ΔfopC strains, have previously been evaluated as potential vaccine candidates against pneumonic tularemia in experimental animals. F. novicida ΔiglB, a Francisella pathogenicity island (FPI) mutant, is deficient in phagosomal escape and intracellular growth, whereas F. novicida ΔfopC, lacking the outer membrane lipoprotein FopC, which is required for evasion of gamma interferon (IFN-γ)-mediated signaling, is able to escape and replicate in the cytosol. To dissect the difference in protective immune mechanisms conferred by these two vaccine strains, we examined the efficacy of the F. novicida ΔiglB and ΔfopC mutants against pulmonary live-vaccine-strain (LVS) challenge and found that both strains provided comparable protection in wild-type, major histocompatibility complex class I (MHC I) knockout, and MHC II knockout mice. However, F. novicida ΔfopC-vaccinated but not F. novicida ΔiglB-vaccinated perforin-deficient mice were more susceptible and exhibited greater bacterial burdens than similarly vaccinated wild-type mice. Moreover, perforin produced by natural killer (NK) cells and release of granzyme contributed to inhibition of LVS replication within macrophages. This NK cell-mediated LVS inhibition was enhanced with anti-F. novicida ΔfopC immune serum, suggesting antibody-dependent cell-mediated cytotoxicity (ADCC) in F. novicida ΔfopC-mediated protection. Overall, this study provides additional immunological insight into the basis for protection conferred by live attenuated F. novicida strains with different phenotypes and supports further investigation of this organism as a vaccine platform for tularemia.     

Intranasal vaccination with a defined attenuated Francisella novicida strain induces gamma interferon-dependent antibody-mediated protection against tularemia

Francisella tularensis is an intracellular gram-negative bacterium that is the causative agent of tularemia and a potential bioweapon. We have characterized the efficacy of a defined F. novicida mutant (DeltaiglC) as a live attenuated vaccine against subsequent intranasal challenge with the wild-type organism. Animals primed with the F. novicida DeltaiglC (KKF24) mutant induced robust splenic gamma interferon (IFN-gamma) and interleukin-12 (IL-12) recall responses with negligible IL-4 production as well as the production of antigen-specific serum immunoglobulin G1 (IgG1) and IgG2a antibodies. BALB/c mice vaccinated intranasally (i.n.) with KKF24 and subsequently challenged with wild-type F. novicida (100 and 1,000 50% lethal doses) were highly protected (83% and 50% survival, respectively) from the lethal challenges. The protection conferred by KKF24 vaccination was shown to be highly dependent on endogenous IFN-gamma production and also was mediated by antibodies that could be adoptively transferred to naive B-cell-deficient mice by inoculation of immune sera. Collectively, the results demonstrate that i.n. vaccination with KKF24 induces a vigorous Th1-type cytokine and antibody response that is protective against subsequent i.n. challenge with the wild-type strain. This is the first report of a defined live attenuated strain providing protection against the inhalation of F. novicida.     

Identification of an orphan response regulator required for the virulence of Francisella spp. and transcription of pathogenicity island genes

Francisella tularensis is a category A agent of biowarfare/biodefense. Little is known about the regulation of virulence gene expression in Francisella spp. Comparatively few regulatory factors exist in Francisella, including those belonging to two-component systems (TCS). However, orphan members of typical TCS can be identified. To determine if orphan TCS members affect Francisella gene expression, a gene encoding a product with high similarity to the Salmonella PmrA response regulator (FTT1557c/FNU0663.2) was deleted in Francisella novicida (a model organism for F. tularensis). The F. novicida pmrA mutant was defective in survival/growth within human and murine macrophage cell lines and was 100% defective in virulence in mice at a dose of up to 10(8) CFU. In addition, the mutant strain demonstrated increased susceptibility to antimicrobial peptide killing, but no differences were observed between the lipid A of the mutant and the parental strain, as has been observed with pmrA mutants of other microbes. The F. novicida pmrA mutant was 100% protective as a single-dose vaccine when challenge was with 10(6) CFU of F. novicida but did not protect against type A Schu S4 wild-type challenge. DNA microarray analysis identified 65 genes regulated by PmrA. The majority of these genes were located in the region surrounding pmrA or within the Francisella pathogenicity island (FPI). These FPI genes are also regulated by MglA, but MglA does not regulate pmrA, nor does PmrA regulate MglA. Thus, the orphan response regulator PmrA is an important factor in controlling virulence in F. novicida, and a pmrA mutant strain is an effective vaccine against homologous challenge.     

The immunologically distinct O antigens from Francisella tularensis subspecies tularensis and Francisella novicida are both virulence determinants and protective antigens

We have determined the sequence of the gene cluster encoding the O antigen in Francisella novicida and compared it to the previously reported O-antigen cluster in Francisella tularensis subsp. tularensis. Immunization with purified lipopolysaccharide (LPS) from F. tularensis subsp. tularensis or F. novicida protected against challenge with Francisella tularensis subsp. holarctica and F. novicida, respectively. The LPS from F. tularensis subsp. tularensis did not confer protection against challenge with F. novicida, and the LPS from F. novicida did not confer protection against challenge with F. tularensis subsp. holarctica. Allelic replacement mutants of F. tularensis subsp. tularensis or F. novicida which failed to produce O antigen were attenuated, but exposure to these mutants did not induce a protective immune response. The O antigen of F. tularensis subsp. tularensis appeared to be important for intracellular survival whereas the O antigen of F. novicida appeared to be critical for serum resistance and less important for intracellular survival.     

Infection-Immunity in Tularemia: Specificity of Cellular Immunity

The relationship between hypersensitivity and cellular resistance to infection with facultative intracellular parasites was studied in mice by using infection-immunity in tularemia as a model system. Delayed hypersensitivity to antigenic fractions of Francisella tularensis was first detected 6 to 7 days after immunization with viable F. tularensis vaccine, at which time immunity against challenge infection developed. Both immunity and delayed-type sensitivity reached maximal levels by 9 to 10 days. Immediate hypersensitivity occurred after immunization with both viable and nonviable tularemia vaccines but could not be correlated with resistance since nonviable antigens were not protective. Attempts to relate resistance to F. tularensis with nonspecific immunity factors were unsuccessful. Immunization of mice with BCG vaccine stimulated protection against infection with F. novicida and Salmonella typhimurium but provided no protection against infection with F. tularensis. Moreover, viable tularemia vaccine, while inducing marked protection against challenge with specific organisms, afforded no protection against infection with S. typhimurium or S. enteritidis. It is concluded that cellular immunity in tularemia involves an immunologically specific component.     

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.     

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.     

Intranasal vaccination induces protective immunity against intranasal infection with virulent Francisella tularensis biovar A

The inhalation of Francisella tularensis biovar A causes pneumonic tularemia associated with high morbidity and mortality rates in humans. Exposure to F. tularensis usually occurs by accident, but there is increasing awareness that F. tularensis may be deliberately released in an act of bioterrorism or war. The development of a vaccine against pneumonic tularemia has been limited by a lack of information regarding the mechanisms required to protect against this disease. Vaccine models for F. tularensis in inbred mice would facilitate investigations of the protective mechanisms and significantly enhance vaccine development. Intranasal vaccination with the attenuated live vaccine strain (LVS) of F. tularensis reproducibly protected BALB/c mice, but not C57BL/6 mice, against intranasal and subcutaneous challenges with a virulent clinical isolate of F. tularensis biovar A (NMFTA1). The resistance of LVS-vaccinated BALB/c mice to intranasal NMFTA1 challenge was increased 100-fold by boosting with live NMFTA1 but not with LVS. The protective response was specific for F. tularensis and required both CD4 and CD8 T cells. The vaccinated mice appeared outwardly healthy for more than 2 months after NMFTA1 challenge, even though NMFTA1 was recovered from more than half of the vaccinated mice. These results show that intranasal vaccination induces immunity that protects BALB/c mice from intranasal infection by F. tularensis biovar A.     

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.     

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.     

Tryptophan prototrophy contributes to Francisella tularensis evasion of gamma interferon-mediated host defense

Francisella tularensis is able to survive and replicate within host macrophages, a trait that is associated with the high virulence of this bacterium. The trpAB genes encode the enzymes required for the final two steps in tryptophan biosynthesis, with TrpB being responsible for the conversion of indole to tryptophan. Consistent with this function, an F. tularensis subsp. novicida trpB mutant is unable to grow in defined medium in the absence of tryptophan. The trpB mutant is also attenuated for virulence in a mouse pulmonary model of tularemia. However, the trpB mutant remains virulent in gamma interferon receptor-deficient (IFN-γR(-/-)) mice, demonstrating that IFN-γ-mediated signaling contributes to clearance of the trpB mutant. IFN-γ limits intracellular survival of the trpB mutant within bone marrow-derived macrophages from wild-type but not IFN-γR(-/-) mice. An F. tularensis subsp. tularensis trpB mutant is also attenuated for virulence in mice and survival within IFN-γ-treated macrophages, indicating that tryptophan prototrophy is also important in a human-virulent F. tularensis subspecies. These results demonstrate that trpB contributes to F. tularensis virulence by enabling intracellular growth under IFN-γ-mediated tryptophan limitation.     

Francisella tularensis subsp. tularensis Schu S4 disulfide bond formation protein B, but not an RND-type efflux pump, is required for virulence

Francisella tularensis subsp. tularensis is a highly virulent bacterium that is a CDC select agent. Despite advancements in the understanding of its biology, details pertaining to virulence are poorly understood. In previous work, we identified a transposon insertion mutant in the FTT0107c locus that was defective in intracellular survival in HepG2 and J77A.1 cells. Here, we report that this mutant was also highly attenuated in vivo. The FTT0107c locus is predicted to encode an ortholog of the disulfide bond formation B protein (DsbB). This designation was confirmed by complementation of an Escherichia coli dsbB mutant. This dsbB mutant of Schu S4 was highly attenuated in mice, but unlike what has been reported for Francisella novicida, intranasal immunization with a sublethal dose did not induce protection against wild-type challenge. dsbB was found to be transcribed in an operon with acrA and acrB, which encode an RND-type efflux pump. However, this pump did not make a significant contribution to virulence because strains with nonpolar deletions in acrA and acrB behaved like wild-type strain Schu S4 with respect to intracellular growth and in vivo virulence. This result is in contrast to a report that an acrB mutant of a live vaccine strain of F. tularensis has decreased virulence in mice. Overall, these results demonstrate key differences between the virulence requirements of Schu S4 and less virulent subspecies of Francisella. We have shown that DsbB is a key participant in intracellular growth and virulence, and our results suggest that there are critical virulence factors that contain disulfide bonds.     

Genome-wide identification of Francisella tularensis virulence determinants

Francisella tularensis is a gram-negative pathogen that causes life-threatening infections in humans and has potential for use as a biological weapon. The genetic basis of the F. tularensis virulence is poorly understood. This study screened a total of 3,936 transposon mutants of the live vaccine strain for infection in a mouse model of respiratory tularemia by signature-tagged mutagenesis. We identified 341 mutants attenuated for infection in the lungs. The transposon disruptions were mapped to 95 different genes, virtually all of which are also present in the genomes of other F. tularensis strains, including human pathogenic F. tularensis strain Schu S4. A small subset of these attenuated mutants carried insertions in the genes encoding previously known virulence factors, but the majority of the identified genes have not been previously linked to F. tularensis virulence. Among these are genes encoding putative membrane proteins, proteins associated with stress responses, metabolic proteins, transporter proteins, and proteins with unknown functions. Several attenuated mutants contained disruptions in a putative capsule locus which partially resembles the poly-gamma-glutamate capsule biosynthesis locus of Bacillus anthracis, the anthrax agent. Deletional mutation analysis confirmed that this locus is essential for F. tularensis virulence.     

Biochemical and immunological properties of ribonucleic acid-rich extracts from Francisella tularensis.

Ribonucleic acid (RNA)-rich extracts derived from the attenuated strain of Francisella tularensis (strain LVS) protected Swiss mice against lethal challenge with F. tularensis strain 425 but not against strain SCHU S4. No killed preparation, including an RNA-rich extract from SCHU S4 itself, offered protection against strain SCHU S4 in contrast to the high level of protection offered against this strain by vaccination with live strain LVS. The protective activity observed against strain 425 was sensitive to ribonuclease but not to Pronase. Protective activity is not a general property of bacterial RNA, since RNA-rich extracts from Staphylococcus aureus offered no protection against tularemia, although disc gel electrophoresis showed similar kinds and amounts of RNA in preparations form F. tularensis and S. aureus. Furthermore, inability to localize activity to a specific region in sucrose gradients suggests a structural rather than an informational role for the RNA in such extracts. RNA-rich extracts from F. tularensis but not from S. aureus were efficient inducers of F. tularensis opsonins in mouse serum, suggesting one mechanism by which such extracts confer protection.     

A defined O-antigen polysaccharide mutant of Francisella tularensis live vaccine strain has attenuated virulence while retaining its protective capacity

Francisella tularensis, the causative agent of tularemia, has been designated a CDC category A select agent because of its low infective dose (<10 CFU), its ready transmission by aerosol, and its ability to produce severe morbidity and high mortality. The identification and characterization of this organism's virulence determinants will facilitate the development of a safe and effective vaccine. We report that inactivation of the wbtA-encoded dehydratase of the O-antigen polysaccharide (O-PS) locus of the still-unlicensed live vaccine strain of F. tularensis (LVS) results in a mutant (the LVS wbtA mutant) with remarkably attenuated virulence. Western blot analysis and immune electron microscopy studies associate this loss of virulence with a complete lack of surface O-PS expression. A likely mechanism for attenuation is shown to be the transformation from serum resistance in the wild-type strain to serum sensitivity in the mutant. Despite this significant attenuation in virulence, the LVS wbtA mutant remains immunogenic and confers protective immunity on mice against challenge with an otherwise lethal dose of either F. tularensis LVS or a fully virulent clinical isolate of F. tularensis type B. Recognition and characterization of the pivotal role of O-PS in the virulence of this intracellular bacterial pathogen may have broad implications for the creation of a safe and efficacious vaccine.     

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.     

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.