Orchestration of the protective immune response to intracellular bacteria: Francisella tularensis as a model organism

Abstract Francisella tularensis is used as a model organism in studies of mechanisms behind the induction of a protective T-cell response in the mammalian host. Protective immunity is associated with a CD4 and CD8 T-cell response towards a mosaic of proteins of F. tularensis and due to HLA restriction, each individual selects her own mosaic. No single protein has so far been shown to be immunodominant. Only live F. tularensis affords effective host protection. Subcellular antigen preparations induce only a marginal protective response even when combined with potent adjuvants such as immunostimulating complexes (ISCOMs). In mice, intradermal injection of live F. tularensis but not of killed bacteria results in an early cytokine expression in the infected liver, including interleukin-12, tumor necrosis factor-α, and interferon-γ. This cytokine response seems to be a prerequisite for effective priming of T cells to an array of proteins of F. tularensis to occur.     

Activation of the inflammasome upon Francisella tularensis infection: interplay of innate immune pathways and virulence factors

Tularaemia is a zoonotic disease caused by the facultative intracellular bacterium Francisella tularensis. The virulence of this pathogen depends on its ability to escape into the cytosol of host cells. Pathogens are detected by the innate immune system's pattern recognition receptors which are activated in response to conserved microbial molecules (pathogen-associated molecular patterns). Cytosolic bacteria are sensed intracellularly, often leading to activation of the cysteine protease caspase-1 within a multimolecular complex called the inflammasome. Caspase-1 activation leads to both host cell death and release of pro-inflammatory cytokines in a process called pyroptosis. Here we review the pathway leading to, and the consequences of, inflammasome activation upon F. tularensis infection both in vitro and in vivo. Finally, we discuss recent data on how other innate immune pathways and F. tularensis virulence factors control the activation of the inflammasome during infection.     

Pleiotropic nature of mutation of resistance to 2,3,5-triphenyl- tetrazolium chloride of Francisella tularensis]

Natural strains of F. tularensis were characterized by sensitivity to 2,3,5-triphenyl tetrazolium chloride (TTC). Development of TTC resistance in the cells of F. tularensis was accompanied by changes in the biological properties of the culture, i.e. the colony morphology, antigenic structure, virulence and immunogenicity for laboratory animals. Moreover, there was a direct correlation between the levels of TTC resistance and resistance to chloramphenicol, erythromycin, tetracycline, furazolidone and rifampicin. The antibiotic resistant mutants of F. tularensis were in turn more resistant to TTC than the initial strains. This could be useful in isolation of polymarked strains of F. tularensis for genetic studies and investigation of the nature of the phenomenon of virulence in F. tularensis.     

Drosophila melanogaster as a model for elucidating the pathogenicity of Francisella tularensis

Drosophila melanogaster is a widely used model organism for research on innate immunity and serves as an experimental model for infectious diseases. The aetiological agent of the zoonotic disease tularaemia, Francisella tularensis, can be transmitted by ticks and mosquitoes and Drosophila might be a useful, genetically amenable model host to elucidate the interactions between the bacterium and its arthropod vectors. We found that the live vaccine strain of F. tularensis was phagocytosed by Drosophila and multiplied in fly haemocytes in vitro and in vivo. Bacteria injected into flies resided both inside haemocytes and extracellularly in the open circulatory system. A continuous activation of the humoral immune response, i.e. production of antimicrobial peptides under control of the imd/Relish signalling pathway, was observed and it may have contributed to the relative resistance to F. tularensis as flies defective in the imd/Relish pathway died rapidly. Importantly, bacterial strains deficient for genes of the F. tularensis intracellular growth locus or the macrophage growth locus were attenuated in D. melanogaster. Our results demonstrate that D. melanogaster is a suitable model for the analysis of interactions between F. tularensis and its arthropod hosts and that it can also be used to identify F. tularensis virulence factors relevant for mammalian hosts.     

Deletion of IglH in virulent Francisella tularensis subsp. holarctica FSC200 strain results in attenuation and provides protection against the challenge with the parental strain

Francisella tularensis, the causative agent of tularemia, is a highly infectious intracellular pathogen with no licensed vaccine available today. The recent search for genome sequences involved in F. tularensis virulence mechanisms led to the identification of the 30-kb region defined as a Francisella pathogenicity island (FPI). In our previous iTRAQ study we described the concerted upregulation of some FPI proteins in different F. tularensis strains cultivated under stress conditions. Among them we identified the IglH protein whose role in Francisella virulence has not been characterized yet. In this work we deleted the iglH gene in a European clinical isolate of F. tularensis subsp. holarctica FSC200. We showed that the iglH gene is necessary for intracellular growth and escape of F. tularensis from phagosomes. We also showed that the iglH mutant is avirulent in a mouse model of infection and persists in the organs for about three weeks after infection. Importantly, mice vaccinated by infection with the iglH mutant were protected against subcutaneous challenge with the fully virulent parental FSC200 strain. This is the first report of a defined subsp. holarctica FPI deletion strain that provides protective immunity against subsequent subcutaneous challenge with a virulent isolate of F. tularensis subsp. holarctica.     

Prophylactic and therapeutic use of antibodies for protection against respiratory infection with Francisella tularensis

The role of Abs in protection against respiratory infection with the intracellular bacterium Francisella tularensis is not clear. To investigate the ability of Abs to clear bacteria from the lungs and prevent systemic spread, immune serum was passively administered i.p. to naive mice before intranasal F. tularensis live vaccine strain infection. It was found that immune serum treatment provided 100% protection against lethal challenge while normal serum or Ig-depleted immune serum provided no protection. Protective efficacy was correlated with increased clearance of bacteria from the lung and required expression of FcgammaR on phagocytes, including macrophages and neutrophils. However, complement was not required for protection. In vitro experiments demonstrated that macrophages were more readily infected by Ab-opsonized bacteria but became highly efficient in killing upon activation by IFN-gamma. Consistent with this finding, in vivo Ab-mediated protection was found to be dependent upon IFN-gamma. SCID mice were not protected by passive Ab transfer, suggesting that T cells but not NK cells serve as the primary source for IFN-gamma. These data suggest that a critical interaction of humoral and cellular immune responses is necessary to provide sterilizing immunity against F. tularensis. Of considerable interest was the finding that serum Abs were capable of conferring protection against lethal respiratory tularemia when given 24-48 h postexposure. Thus, this study provides the first evidence for the therapeutic use of Abs in Francisella-infected individuals.     

Comparative analysis of the immune response of a rabbit to antigens to live and killed Francisella species bacteria]

Serum antibodies were analyzed in rabbits immunized with live and formalin-killed Francisella (F. tularensis, F. novicida, F. novicida-like, and F. philomiragia). Passive hemagglutination test with erythrocytes sensitized by these bacteria' LPS showed much higher titers of species-specific antibodies in all sera to live microorganisms than sera to killed bacteria. The results of immunoblotting with purified LPS and bacterial lysates indicate that sera to live bacteria contained mainly immunoglobulins to species-specific antigenic epitopes of LPS O-polysaccharide chain and few antibodies to the protein component of the cell. By contrast, killed bacterial cells induced weak production of antibodies to S-LPS and a pronounced antibody response to protein antigens. Besides the quantitative differences, live and killed bacteria differed by the qualitative spectrum of immunodominant proteins. Serum to live F. tularensis 15/10 contained antibodies to at least 3 immunodominant antigens of the cell, while serum to killed bacteria contained antibodies to only two of these. Immunoglobulins to protein antigens, absent in homologous sera to live bacteria, were detected in the sera to killed F. novicida and F. novicida-like bacteria. Both sera to F. philomiragia had antibodies reacting with LPS epitopes and immunodominant complex containing protein. In contrast to other Francisella, F. philomiragia was found to synthesize an uncommon LPS representing two major lipooligosaccharides with different molecular weights and antigenic specificity. Therefore, immune response of the host to live and killed Francisella is different: live cells more effectively induce the production of antibodies to S-LPS epitopes, while killed ones to protein antigens.     

A new cell assay to determine the virulence of Francisella tularensis

A cell culture assay to determine the virulence of Francisella tularensis was devised. Murine cell lines P388 and J774 were significantly more susceptible to F. tularensis Schu4 than the attenuated live vaccine strain. The ability of F. tularensis strains to cause cell death correlated with their virulence to mice. Use of this assay with infected cells separated from susceptible uninfected cells by a membrane with 0.1 μm pores, failed to demonstrate possible diffusible exotoxins produced by F. tularensis.     

Role of two outer membrane antigens in the induction of protective immunity against Francisella tularensis strains of different virulence

Abstract A crude outer membrane preparation from Francisella tularensis live vaccine strain was used to immunise mice. Immunised mice were completely protected from a F. tularensis challenge. We evaluated the role of two major outer membrane antigens in the induction of protective immunity, namely lipopolysaccharide and an outer membrane protein FopA . We presented FopA to the immune system using an aromatic amino acid dependent Salmonella typhimurium as a vector. Although mice mounted an immune response to cloned FopA no significant protection was induced. However, lipopolysaccharide-immunised mice were completely protected from a F. tularensis live vaccine strain challenge. No increase in LD₅₀ was observed using F. tularensis Schu4 as the challenge strain, although there was a significant increase in time to death. These data question the validity of the murine F. tularensis live vaccine strain model.     

Francisella tularensis selectively induces proinflammatory changes in endothelial cells

Naturally acquired infections with Francisella tularensis, the bacterial agent of tularemia, occur infrequently in humans. However, the high infectivity and lethality of the organism in humans raise concerns that it might be exploited as a weapon of bioterrorism. Despite this potential for illicit use, the pathogenesis of tularemia is not well understood. To examine how F. tularensis interacts with cells of its mammalian hosts, we tested the ability of a live vaccine strain (LVS) to induce proinflammatory changes in cultured HUVEC. Living F. tularensis LVS induced HUVEC to express the adhesion molecules VCAM-1 and ICAM-1, but not E-selectin, and to secrete the chemokine CXCL8, but not CCL2. Stimulation of HUVEC by the living bacteria was partially suppressed by polymyxin B, an inhibitor of LPS, but did not require serum, suggesting that F. tularensis LVS does not stimulate endothelium through the serum-dependent pathway that is typically used by LPS from enteric bacteria. In contrast to the living organisms, suspensions of killed F. tularensis LVS acquired the ability to increase endothelial expression of both E-selectin and CCL2. Up-regulation of E-selectin and CCL2 by the killed bacteria was not inhibited by polymyxin B. Exposure of HUVEC to either live or killed F. tularensis LVS for 24 h promoted the transendothelial migration of subsequently added neutrophils. These data indicate that multiple components of F. tularensis LVS induce proinflammatory changes in endothelial cells in an atypical manner that may contribute to the exceptional infectivity and virulence of this pathogen.     

Species- and genus-specific antigenic epitopes of Francisella tularensis lipopolysaccharides

Lipopolysaccharide (LPS) antigenic epitopes of natural virulent and isogenic avirulent Francisella tularensis strains and other species of the Francisella genus (F. novicida, F. novicida-like, and F. philomiragia) were studied by dot and immunoblotting. Polyclonal rabbit and human sera to virulent F. tularensis strains and monoclonal antibodies to F. tularensis LPS O-side chain were used for detecting species- and genus-specific LPS epitopes. Typical virulent F. tularensis strains produce two types of S-LPS with different antigenic specificity simultaneously. Antigenic determinants of two LPS types were located in LPS O-polysaccharide but not in the core oligosaccharide. The epitopes of the first LPS type were characterized by species specificity for F. tularensis in contrast to determinants of the second LPS type, which had epitopes common with F. novicida. Cross exhaustion of human and rabbit antitularemic sera by F. tularensis and F. novicida LPS showed that F. novicida LPS molecules contained at least two epitopes--highly specific for F. novicida and common with the second type of F. tularensis LPS. The immune response of rabbits and humans to F. tularensis LPS epitopes was different in principle. Sera from rabbits immunized with vaccine and virulent F. tularensis strains contained antibodies "recognizing" antigenic epitopes of two S-LPS forms of the bacterium: type 1 species-specific (in high titers) and type 2 epitopes common with F. novicida LPS (in low titers). In addition to these, sera from patients with tularemia contain immunoglobulins to species-specific epitopes of F. novicida LPS in high titers. Experiments on avirulent mutants showed that in some cases attenuation of F. tularensis can involve loss of species-specific LPS form, while S-LPS with epitopes common with F. novicida LPS will be retained. The difference in specificity of human and rabbit antitularemic antibodies is due to individual features in the host immune system.     

Mechanisms of vaccine-induced protective immunity against Coxiella burnetii infection in BALB/c mice

To elucidate the mechanisms of vaccine-induced protective immunity against Coxiella burnetii infection, we compared the protective efficacy and immunogenicity between formalin-inactivated phase I vaccine (PI-V) and phase II vaccine (PII-V) in BALB/c mice. PI-V generated significant protection while PII-V did not confer measurable protection. Analysis of cytokine and subclass Ab responses indicated that both PI-V and PII-V were able to induce a Th1-dominant immune response but did not identify the component of host response that distinguished their ability to induce protective immunity. Interestingly, immunoblot analysis identified a difference between PI-V and PII-V vaccinates in antigenic recognition by specific Ab isotypes. The observation that PI-LPS elicited significant protection but PII-LPS did not confer measurable protection suggests PI-LPS may play a key role in PI-V-induced protection. Adoptive transfer of either immune sera or splenocytes mediated significant protection in naive BALB/c mice, supporting the notion that both humoral and cellular immunity are important for development of protective immunity. However, the evidence that immune sera and B cells were unable to control infection while T cells conferred significant protection in SCID mice supports the hypothesis that T cell-mediated immunity is critical for host defense against C. burnetii infection. This report presents novel evidence to highlight the importance of PI-LPS and Abs in protective immunity and has important implications for the design of new generation vaccines against Q fever.     

Live tularemia vaccine confers protection against lethal Legionella and Listeria infections in experimental animals

Abstract The efficacy of a live Francisella tularensis vaccine strain to cause nonspecific immunity toward experimental legionellosis and listeriosis was studied. Immunisation with tularemia vaccine protected over 80% and 17% of experimental animals against subsequent lethal challenge with Legionella pneumophila and Listeria monocytogenes , respectively. The protection was maximal during the first month following immunisation and declined thereafter. In order to delineate the immunostimulatory moieties of the Francisella microbe, several cell wall proteins have been purified and characterized. However, isolated cell wall components failed to induce protection.     

Low dose vaccination with attenuated Francisella tularensis strain SchuS4 mutants protects against tularemia independent of the route of vaccination

Tularemia, caused by the gram-negative bacterium Francisella tularensis, is a severe, sometimes fatal disease. Interest in tularemia has increased over the last decade due to its history as a biological weapon. In particular, development of novel vaccines directed at protecting against pneumonic tularemia has been an important goal. Previous work has demonstrated that, when delivered at very high inoculums, administration of live, highly attenuated strains of virulent F. tularensis can protect against tularemia. However, lower vaccinating inoculums did not offer similar immunity. One concern of using live vaccines is that the host may develop mild tularemia in response to infection and use of high inoculums may contribute to this issue. Thus, generation of a live vaccine that can efficiently protect against tularemia when delivered in low numbers, e.g. <100 organisms, may address this concern. Herein we describe the ability of three defined, attenuated mutants of F. tularensis SchuS4, deleted for FTT0369c, FTT1676, or FTT0369c and FTT1676, respectively, to engender protective immunity against tularemia when delivered at concentrations of approximately 50 or fewer bacteria. Attenuated strains for use as vaccines were selected by their inability to efficiently replicate in macrophages in vitro and impaired replication and dissemination in vivo. Although all strains were defective for replication in vitro within macrophages, protective efficacy of each attenuated mutant was correlated with their ability to modestly replicate and disseminate in the host. Finally, we demonstrate the parenteral vaccination with these strains offered superior protection against pneumonic tularemia than intranasal vaccination. Together our data provides proof of principle that low dose attenuated vaccines may be a viable goal in development of novel vaccines directed against tularemia.     

Superoxide dismutase B gene (sodB)-deficient mutants of Francisella tularensis demonstrate hypersensitivity to oxidative stress and attenuated virulence

A Francisella tularensis live vaccine strain mutant (sodB(Ft)) with reduced Fe-superoxide dismutase gene expression was generated and found to exhibit decreased sodB activity and increased sensitivity to redox cycling compounds compared to wild-type bacteria. The sodB(Ft) mutant also was significantly attenuated for virulence in mice. Thus, this study has identified sodB as an important F. tularensis virulence factor.     

Virulence determinants and protective antigens of Francisella tularensis

Very little is known about virulence mechanisms of the highly virulent bacterium Francisella tularensis. Specific genetic features of F. tularensis have been obstacles for the development of effective tools for genetic manipulation. However, recent genomic sequencing and large-scale proteomic work have resulted in a substantial increase in the knowledge of F. tularensis. There is also a paucity of information on potential vaccine candidates. Recent work assessing the protective efficacy of the F. tularensis lipopolysaccharide has resulted in important contributions to the understanding of host-protective mechanisms. T-cell-mediated immunity appears to be crucial to protect against virulent F. tularensis strains. Few other vaccine candidates have been identified.     

Host-pathogen o-methyltransferase similarity and its specific presence in highly virulent strains of Francisella tularensis suggests molecular mimicry

Whole genome comparative studies of many bacterial pathogens have shown an overall high similarity of gene content (>95%) between phylogenetically distinct subspecies. In highly clonal species that share the bulk of their genomes subtle changes in gene content and small-scale polymorphisms, especially those that may alter gene expression and protein-protein interactions, are more likely to have a significant effect on the pathogen's biology. In order to better understand molecular attributes that may mediate the adaptation of virulence in infectious bacteria, a comparative study was done to further analyze the evolution of a gene encoding an o-methyltransferase that was previously identified as a candidate virulence factor due to its conservation specifically in highly pathogenic Francisella tularensis subsp. tularensis strains. The o-methyltransferase gene is located in the genomic neighborhood of a known pathogenicity island and predicted site of rearrangement. Distinct o-methyltransferase subtypes are present in different Francisella tularensis subspecies. Related protein families were identified in several host species as well as species of pathogenic bacteria that are otherwise very distant phylogenetically from Francisella, including species of Mycobacterium. A conserved sequence motif profile is present in the mammalian host and pathogen protein sequences, and sites of non-synonymous variation conserved in Francisella subspecies specific o-methyltransferases map proximally to the predicted active site of the orthologous human protein structure. Altogether, evidence suggests a role of the F. t. subsp. tularensis protein in a mechanism of molecular mimicry, similar perhaps to Legionella and Coxiella. These findings therefore provide insights into the evolution of niche-restriction and virulence in Francisella, and have broader implications regarding the molecular mechanisms that mediate host-pathogen relationships.     

Active suppression of the pulmonary immune response by Francisella tularensis Schu4

Francisella tularensis is an obligate, intracellular bacterium that causes acute, lethal disease following inhalation. As an intracellular pathogen F. tularensis must invade cells, replicate, and disseminate while evading host immune responses. The mechanisms by which virulent type A strains of Francisella tularensis accomplish this evasion are not understood. Francisella tularensis has been shown to target multiple cell types in the lung following aerosol infection, including dendritic cells (DC) and macrophages. We demonstrate here that one mechanism used by a virulent type A strain of F. tularensis (Schu4) to evade early detection is by the induction of overwhelming immunosuppression at the site of infection, the lung. Following infection and replication in multiple pulmonary cell types, Schu4 failed to induce the production of proinflammatory cytokines or increase the expression of MHCII or CD86 on the surface of resident DC within the first few days of disease. However, Schu4 did induce early and transient production of TGF-beta, a potent immunosuppressive cytokine. The absence of DC activation following infection could not be attributed to the apoptosis of pulmonary cells, because there were minimal differences in either annexin or cleaved caspase-3 staining in infected mice compared with that in uninfected controls. Rather, we demonstrate that Schu4 actively suppressed in vivo responses to secondary stimuli (LPS), e.g., failure to recruit granulocytes/monocytes and stimulate resident DC. Thus, unlike attenuated strains of F. tularensis, Schu4 induced broad immunosuppression within the first few days after aerosol infection. This difference may explain the increased virulence of type A strains compared with their more attenuated counterparts.     

Influence of nutrient status and grazing pressure on the fate of Francisella tularensis in lake water

The natural reservoir of Francisella tularensis , the causative agent of tularaemia, is yet to be identified. We investigated the possibility that Francisella persists in natural aquatic ecosystems between outbreaks. It was hypothesized that nutrient-rich environments, with strong protozoan predation, favour the occurrence of the tularaemia bacterium. To investigate the differences in adaptation to aquatic environments of the species and subspecies of Francisella , we screened 23 strains for their ability to survive grazing by the ciliate Tetrahymena pyriformis . All the Francisella strains tested were consumed at a low rate, although significant differences between subspecies were found. The survival and virulence of gfp -labelled F. tularensis ssp. holarctica were then studied in a microcosm experiment using natural lake water, with varying food web complexities and nutrient availabilities. High nutrient conditions in combination with high abundances of nanoflagellates were found to favour F. tularensis ssp. holarctica . The bacterium was observed both free-living and within the cells of a nanoflagellate. Francisella tularensis entered a viable but nonculturable state during the microcosm experiment. When studied over a longer period of time, F. tularensis ssp. holarctica survived in the lake water, but loss of virulence was not prevented by either high nutrient availability or the presence of predators.