Proteomic analysis of bronchoalveolar lavage fluid proteins from mice infected with Francisella tularensis ssp. novicida

Francisella tularensis causes the zoonosis tularemia in humans and is one of the most virulent bacterial pathogens. We utilized a global proteomic approach to characterize protein changes in bronchoalveolar lavage fluid from mice exposed to one of three organisms, F. tularensis ssp. novicida, an avirulent mutant of F. tularensis ssp. novicida (F.t. novicida-ΔmglA), and Pseudomonas aeruginosa. The composition of bronchoalveolar lavage fluid (BALF) proteins was altered following infection, including proteins involved in neutrophil activation, oxidative stress, and inflammatory responses. Components of the innate immune response were induced including the acute phase response and the complement system; however, the timing of their induction varied. F. tularensis ssp. novicida infected mice do not appear to have an effective innate immune response in the first hours of infection; however, within 24 h, they show an upregulation of innate immune response proteins. This delayed response is in contrast to P. aeruginosa infected animals which show an early innate immune response. Likewise, F.t. novicida-ΔmglA infection initiates an early innate immune response; however, this response is diminished by 24 h. Finally, this study identifies several candidate biomarkers, including Chitinase 3-like-1 (CHI3L1 or YKL-40) and peroxiredoxin 1, that are associated with F. tularensis ssp. novicida but not P. aeruginosa infection.     

Genetic elements for selection, deletion mutagenesis and complementation in Francisella spp

Francisella novicida is a gram-negative pathogen that can induce disease in mice that mimics human tularemia, and is nearly identical to Francisella tularensis at the genomic level. In this work a number of antibiotic marker cassettes that incorporate a strong F. novicida promoter is constructed, which greatly enhances selection in F. novicida and F. tularensis. Two low-copy plasmid vectors based on a broad-host-range plasmid, and an integrating vector have also been made, and these can be used for genetic complementation. Two general approaches to deletion mutagenesis in F. novicida is also described.     

Genetic dissection of the Francisella novicida restriction barrier

Francisella tularensis is the causative agent of tularemia and is a category A select agent. Francisella novicida, considered by some to be one of four subspecies of F. tularensis, is used as a model in pathogenesis studies because it causes a disease similar to tularemia in rodents but is not harmful to humans. F. novicida exhibits a strong restriction barrier which reduces the transformation frequency of foreign DNA up to 10(6)-fold. To identify the genetic basis of this barrier, we carried out a mutational analysis of restriction genes identified in the F. novicida genome. Strains carrying combinations of insertion mutations in eight candidate loci were created and assayed for reduced restriction of unmodified plasmid DNA introduced by transformation. Restriction was reduced by mutations in four genes, corresponding to two type I, one type II, and one type III restriction system. Restriction was almost fully eliminated in a strain in which all four genes were inactive. The strongest contributor to the restriction barrier, the type II gene, encodes an enzyme which specifically cleaves Dam-methylated DNA. Genome comparisons show that most restriction genes in the F. tularensis subspecies are pseudogenes, explaining the unusually strong restriction barrier in F. novicida and suggesting that restriction was lost during evolution of the human pathogenic subspecies. As part of this study, procedures were developed to introduce unmodified plasmid DNA into F. novicida efficiently, to generate defined multiple mutants, and to produce chromosomal deletions of multiple adjacent genes.     

Phase variations of Francisella tularensis lipopolysaccharide in human infection and immunization

The comparative study of the specificity of antibodies in human sera after tularemia infection and immunization with live tularemia infection was carried out with the use of passive hemagglutination and immunoblotting techniques. The sera of tularemia patients contained two different types of immunoglobulins: strictly specific to the antigenic epitopes of F. tularensis Iipopolysaccharide (LPS) and strictly specific to F. tularensis subsp. novicida LPS. Such phenomenon may be due to phase variations of the antigenic structure of F. tularensis LPS in the body of a slightly susceptible host. The immune sera of vaccinated were found to contain antibodies, strictly specific only to F. tularensis LPS. At the same time in one vaccinee by the presence of pronounced postvaccinal reactions was found sharply defined interaction between serum imunoglobulins and F. tularensis subsp. novicida LPS. As the result, the data on the possibility of the antigenic modification of F. tularensis in tularemia infection in humans were obtained. At the same time antigenic epitopes, characteristic of faintly pathogenic and closely related F. tularensis novicida LPS, appeared in the structure of F. tularensis LPS.     

Respiratory infection with Francisella novicida induces rapid dystrophic cardiac calcinosis (DCC)

Francisella tularensis causes pulmonary tularemia and death in humans when left untreated. Here, using a novel aerosol infection model, we show that acute pulmonary Francisella novicida infection not only causes pneumonia and liver damage, but also induces dystrophic cardiac calcinosis (DCC) in BALB/c mice. C57BL/6 mice also develop pneumonia and hepatic damage, but fail to develop DCC. Development of DCC in BALB/c mice is associated with significant induction of RANKL but not osteopontin in their organs. Depletion of lung macrophages prior to infection markedly reduces pericarditis and calcification in BALB/c mice but does not increase their susceptibility to infection.     

Genetic diversity within the genus Francisella as revealed by comparative analyses of the genomes of two North American isolates from environmental sources

ABSTRACT: BACKGROUND: Francisella tularensis is an intracellular pathogen that causes tularemia in humans and the public health importance of this bacterium has been well documented in recent history. Francisella philomiragia, a distant relative of F. tularensis, is thought to constitute an environmental lineage along with Francisella novicida. Nevertheless, both F. philomiragia and F. novicida have been associated with human disease, primarily in immune-compromised individuals. To understand the genetic relationships and evolutionary contexts among different lineages within the genus Francisella, the genome of Francisella spp. strain TX07-7308 was sequenced and compared to the genomes of F. philomiragia strains ATCC 25017 and 25015, F. novicida strain U112, and F. tularensis strain Schu S4. RESULTS: The size of strain ATCC 25017 chromosome was 2,045,775 bp and contained 1,983 protein-coding genes. The size of strain TX07-7308 chromosome was 2,035,931 bp and contained 1,980 protein-coding genes. Pairwise BLAST comparisons indicated that strains TX07-7308 and ATCC 25017 contained 1700 protein coding genes in common. NUCmer analyses revealed that the chromosomes of strains TX07-7308 and ATCC 25017 were mostly collinear except for a few gaps, translocations, and/or inversions. Using the genome sequence data and comparative analyses with other members of the genus Francisella (e.g., F. novicida strain U112 and F. tularensis strain Schu S4), several strain-specific genes were identified. Strains TX07-7308 and ATCC 25017 contained an operon with six open reading frames encoding proteins related to enzymes involved in thiamine biosynthesis that was absent in F. novicida strain U112 and F. tularensis strain Schu S4. Strain ATCC 25017 contained an operon putatively involved in lactose metabolism that was absent in strain TX07-7308, F. novicida strain U112, and F. tularensis strain Schu S4. In contrast, strain TX07-7308 contained an operon putatively involved in glucuronate metabolism that was absent in the genomes of strain ATCC 25017, F. novicida strain U112, and F. tularensis strain Schu S4. The polymorphic nature of polysaccharide biosynthesis/modification gene clusters among different Francisella strains was also evident from genome analyses. CONCLUSIONS: From genome comparisons, it appeared that genes encoding novel functions have contributed to the metabolic enrichment of the environmental lineages within the genus Francisella. The inability to acquire new genes coupled with the loss of ancestral traits and the consequent reductive evolution may be a cause for, as well as an effect of, niche selection of F. tularensis. Sequencing and comparison of the genomes of more isolates are required to obtain further insights into the ecology and evolution of different species within the genus Francisella.     

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.     

Francisella novicida LPS has greater immunobiological activity in mice than F. tularensis LPS,and contributes to F. novicida murine pathogenesis

To further understand the role of LPS in the pathogenesis of Francisella infection, we characterized murine infection with F. novicida, and compared immunobiological activities of F. novicida LPS and the LPS from F. tularensis live vaccine strain (LVS). F. novicida had a lower intradermal LD(50) in BALB/cByJ mice than F. tularensis LVS, and mice given a lethal F. novicida dose intraperitoneally died faster than those given the same lethal F. tularensis LVS dose. However, the pattern of in vivo dissemination was similar, and in vitro growth of both bacteria in bone marrow-derived macrophages was comparable. F. novicida LPS stimulated very modest in vitro proliferation of mouse splenocytes at high doses, but F. tularensis LVS LPS did not. Murine bone marrow macrophages treated in vitro with F. novicida LPS produced IL12 and TNF-alpha, but did not produce detectable interferon-gamma, IL10, or nitric oxide; in contrast, murine macrophages treated with F. tularensis LVS LPS produced none of these mediators. In contrast to clear differences in stimulation of proliferation and especially cytokines, both types of purified LPS stimulated early protection against lethal challenge of mice with F. tularensis LVS, but not against lethal challenge with F. novicida. Thus, although LPS recognition may not be a major factor in engendering protection, the ability of F. novicida LPS to stimulate the production of proinflammatory cytokines including TNF-alpha likely contributes to the increased virulence for mice of F. novicida compared to F. tularensis LVS.     

Immunologic consequences of Francisella tularensis live vaccine strain infection: role of the innate immune response in infection and immunity

Francisella tularensis (Ft), a Gram-negative intracellular bacterium, is the etiologic agent of tularemia. Although attenuated for humans, i.p. infection of mice with <10 Ft live vaccine strain (LVS) organisms causes lethal infection that resembles human tularemia, whereas the LD50 for an intradermal infection is >10(6) organisms. To examine the immunological consequences of Ft LVS infection on the innate immune response, the inflammatory responses of mice infected i.p. or intradermally were compared. Mice infected i.p. displayed greater bacterial burden and increased expression of proinflammatory genes, particularly in the liver. In contrast to most LPS, highly purified Ft LVS LPS (10 microg/ml) was found to be only minimally stimulatory in primary murine macrophages and in HEK293T cells transiently transfected with TLR4/MD-2/CD14, whereas live Ft LVS bacteria were highly stimulatory for macrophages and TLR2-expressing HEK293T cells. Despite the poor stimulatory activity of Ft LVS LPS in vitro, administration of 100 ng of Ft LVS LPS 2 days before Ft LVS challenge severely limited both bacterial burden and cytokine mRNA and protein expression in the absence of detectable Ab at the time of bacterial challenge, yet these mice developed a robust IgM Ab response within 2 days of infection and survived. These data suggest that prior administration of Ft LVS LPS protects the host by diminishing bacterial burden and blunting an otherwise overwhelming inflammatory response, while priming the adaptive immune response for development of a strong Ab response.     

Myeloid differentiation factor-88 (MyD88) is essential for control of primary in vivo Francisella tularensis LVS infection, but not for control of intra-macrophage bacterial replication

The means by which Francisella tularensis, the causative agent of tularemia, are recognized by mammalian immune systems are poorly understood. Here we wished to explore the contribution of the MyD88/Toll-like receptor signaling pathway in initiating murine responses to F. tularensis Live Vaccine Strain (LVS). MyD88 knockout (KO) mice, but not TLR2-, TLR4- or TLR9-deficient mice, rapidly succumbed following in vivo bacterial infection via the intradermal route even with a very low dose of LVS (5 x 10(1)) that was 100,000-fold less than the LD(50) of normal wild-type (WT) mice. By day 5 after LVS infection, bacterial organ burdens were 5-6 logs higher in MyD88 knockout mice; further, unlike infected WT mice, levels of interferon-gamma in the sera of LVS-infected MyD88 KO were undetectable. An in vitro culture system was used to assess the ability of bone marrow macrophages derived from either KO or WT mice to support bacterial growth, or to control intracellular bacterial replication when co-cultured with immune lymphocytes. In this assay, bacterial replication was similar in macrophages derived from either WT or any of the TLR KO mice. Bacterial growth was controlled in co-cultures containing macrophages from MyD88 KO mice or TLR KO mice as well as in co-cultures containing immune WT splenic lymphocytes and WT macrophages. Further, MyD88-deficient LVS-immune splenocytes controlled intracellular growth comparably to those from normal mice. Thus MyD88 is essential for innate host resistance to LVS infection, but is not required for macrophage control of intracellular bacterial growth.     

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.     

Modulation of biogenesis of the Francisella tularensis subsp. novicida-containing phagosome in quiescent human macrophages and its maturation into a phagolysosome upon activation by IFN-gamma

Francisella tularensis is a highly virulent facultative intracellular pathogen that has been categorized as a class A bioterrorism agent, and is classified into four subsp, tularensis, holarctica, mediasiatica and novicida. Although the ability of F. tularensis subsp. novicida to cause tularemia in mice is similar to the virulent subsp. tularensis and holarctica, it is attenuated in humans. It is not known whether attenuation of F. tularensis subsp. novicida in humans is resulting from a different route of trafficking within human macrophages, compared with the tularensis or holarctica subsp. Here we show that in quiescent human monocytes-derived macrophages (hMDMs), the F. tularensis subsp. novicida containing phagosome (FCP) matures into a late endosome-like stage that acquires the late endosomal marker LAMP-2 but does not fuse to lysosomes. This modulation of phagosome biogenesis by F. tularensis is followed by disruption of the phagosome at 4-12 h and subsequent bacterial escape into cytoplasm where the organism replicates. In IFN-gamma-activated hMDMs, intracellular replication of F. tularensis is completely inhibited, and is associated with failure of the organism to escape from the phagosome into the cytoplasm for up to 24 h after infection. In IFN-gamma-activated hMDMs, the FCPs acquire the lysosomal enzymes Cathepsin D, which is excluded in quiescent hMDMs. When the lysosomes of IFN-gamma-activated hMDMs are preload with Texas Red Ovalbumin or BSA-gold, the FCPs acquire both lysosomal tracers. In contrast, both lysosomal tracers are excluded from the FCPs within quiescent hMDMs. We conclude that although F. tularensis subsp. novicida is attenuated in humans, it modulates biogenesis of its phagosome into a late endosome-like compartment followed by bacterial escape into the cytoplasm within quiescent hMDMs, similar to the virulent subsp. tularensis. In IFN-gamma-activated hMDMs, the organism fails to escape into the cytoplasm and its phagosome fuses to lysosomes, similar to inert particles.     

Innate and adaptive immune responses to an intracellular bacterium,Francisella tularensis live vaccine strain

The immune response to intracellular bacterium, Francisella tularensis, which causes tularemia and is proposed to be a potential bioterrorism pathogen, has been studied in mice using the attenuated live vaccine strain (LVS). Here we review this infection model, which provides a convenient means of studying protective immune mechanisms not only for Francisella, but also for the large and important class of intracellular pathogens.     

Mast cell TLR2 signaling is crucial for effective killing of Francisella tularensis

TLR signaling is critical for early host defense against pathogens, but the contributions of mast cell TLR-mediated mechanisms and subsequent effector functions during pulmonary infection are largely unknown. We have previously demonstrated that mast cells, through the production of IL-4, effectively control Francisella tularensis replication. In this study, the highly human virulent strain of F. tularensis SCHU S4 and the live vaccine strain were used to investigate the contribution of mast cell/TLR regulation of Francisella. Mast cells required TLR2 for effective bacterial killing, regulation of the hydrolytic enzyme cathepsin L, and for coordination and trafficking of MHC class II and lysosomal-associated membrane protein 2. Infected TLR2(-/-) mast cells, in contrast to wild-type and TLR4(-/-) cells, lacked detectable IL-4 and displayed increased cell death with a 2-3 log increase of F. tularensis replication, but could be rescued with rIL-4 treatment. Importantly, MHC class II and lysosomal-associated membrane protein 2 localization with labeled F. tularensis in the lungs was greater in wild-type than in TLR2(-/-) mice. These results provide evidence for the important effector contribution of mast cells and TLR2-mediated signaling on early innate processes in the lung following pulmonary F. tularensis infection and provide additional insight into possible mechanisms by which intracellular pathogens modulate respiratory immune defenses.     

Construction of targeted insertion mutations in Francisella tularensis subsp. novicida

Francisella tularensis is one of the most deadly bacterial agents, yet most of the genetic determinants of pathogenesis are still unknown. We have developed an efficient targeted mutagenesis strategy in the model organism F. tularensis subsp. novicida by utilizing universal priming of optimized antibiotic resistance cassettes and splicing by overlap extension (SOE). This process enables fast and efficient construction of targeted insertion mutations in F. tularensis subsp. novicida that have characteristics of nonpolar mutations; optimized targeted mutagenesis strategies will promote the study of this mysterious bacterium and facilitate vaccine development against tularemia. Moreover the general strategy of gene disruption by PCR-based antibiotic resistance cassette insertion is broadly applicable to many bacterial species.     

Identification of Francisella species and discrimination of type A and type B strains of F. tularensis by 16S rRNA analysis.

Tularemia is a zoonotic disease, occurring throughout the Northern Hemisphere. The causative agent, the bacterium Francisella tularensis, is represented by two main types. Type A is found in North America, whereas type B is mainly found in Asia and Europe and to a minor extent in North America. No routine technique for rapid diagnosis of tularemia has been generally applied. We have partially sequenced 16S rRNAs of two F. tularensis strains, as well as the closely related Francisella novicida. Of 550 nucleotides analyzed, only one difference in 16S rRNA primary sequence was found. This 16S rRNA analysis enabled the construction of oligonucleotides to be used as genus- and type-specific probes. Such probes were utilized for the establishment of a method for rapid and selective detection of the organism. This method allowed identification of Francisella spp. at the level of genus and also discrimination of type A and type B strains of F. tularensis. The analysis also permitted the detection of F. tularensis in spleen tissue from mice infected with the bacterium. The results presented will enable studies on the epizootiology and epidemiology of Francisella spp.