Galleria mellonella as a model host to study infection by the Francisella tularensis live vaccine strain

We used the killing of Galleria mellonella (Lepidoptera: Pyralidae; the greater wax moth) caterpillar by the live vaccine strain (LVS) of Francisella tularensis to develop an invertebrate host system that can be used to study F. tularensis infection and the in vivo effects of antibacterial compounds on F. tularensis LVS. After injection into the insect hemocoel, F. tularensis LVS, killed caterpillars despite the association of LVS with hemocytes. The rate of killing depended on the number of bacteria injected. Antibiotic therapy with ciprofloxacin, levofloxacin or streptomycin administered before or after inoculation prolonged survival and decreased the tissue burden of F. tularensis in the hemocoel. Delayed drug treatment reduced the efficacy of antibacterials and especially streptomycin. The G. mellonella-F. tularensis LVS system may facilitate the in vivo study of F. tularensis, efficacy with antibacterial agents.     

Role of neutrophils and NADPH phagocyte oxidase in host defense against respiratory infection with virulent Francisella tularensis in mice

Francisella tularensis subspecies (subsp.) tularensis is a CDC Category A biological warfare agent and inhalation of as few as 15 bacilli can initiate severe disease. Relatively little is known about the cellular and molecular mechanisms of host defense against respiratory infection with subsp. tularensis. In this study, we examined the role of neutrophils and NADPH phagocyte oxidase in host resistance to pulmonary infection in a mouse intranasal infection model. We found that despite neutrophil recruitment to the lungs and increased concentrations of neutrophil-chemotactic chemokines (KC, MIP-2 and RANTES) in the bronchoalveolar lavage fluid following intranasal inoculation of the pathogen, neither depletion of neutrophils nor enhancement of their recruitment into the lungs had any impact on bacterial burdens or survival rate/time. Nevertheless, mice deficient in NADPH phagocyte oxidase (gp91(phox?/?)) did exhibit higher tissue and blood bacterial burdens and succumbed to infection one day earlier than wild-type C57BL/6 mice. These results imply that although neutrophils are not a major effector cell in defense against subsp. tularensis infection, NADPH phagocyte oxidase does play a marginal role.     

Transcriptional profiling of Francisella tularensis infected peripheral blood mononuclear cells: a predictive tool for tularemia

In this study, we analyzed temporal gene expression patterns in human peripheral blood mononuclear cells (PBMCs) infected with the Francisella tularensis live vaccine strain from 1 to 24 h utilizing a whole human Affymetrix gene chip. We found that a considerable number of induced genes had similar expression patterns and functions as reported previously for gene expression profiling in patients with ulceroglandular tularemia. Among the six uniquely regulated genes reported for tularemia patients as being part of the alarm signal gene cluster, five, namely caspase 1, PSME2, TAP-1, GBP1, and GCH1, were induced in vitro. We also detected four out of the seven potential biomarkers reported in tularemia patients, namely TNFAIP6 at 4 h and STAT1, TNFSF10, and SECTM1 at 16 and 24 h. These observations underscore the value of using microarray expression profiling as an in vitro tool to identify potential biomarkers for human infection and disease. Our results indicate the potential involvement of several host pathways/processes in Francisella infection, notably those involved in calcium, zinc ion binding, PPAR signaling, and lipid metabolism, which further refines the current knowledge of F. tularensis infection and its effects on the human host. Ultimately, this study provides support for utilizing in vitro microarray gene expression profiling in human PBMCs to identify biomarkers of infection and predict in vivo immune responses to infectious agents.     

Intracellular survival mechanisms of Francisella tularensis, a stealth pathogen

Research on the highly virulent and contagious, facultative intracellular bacterium Francisella tularensis has come into the limelight recently, but still little is known regarding its virulence mechanisms. This review summarizes recent studies on its intramacrophage survival mechanisms, some of which appear to be novel.     

Role of NK cells in host defense against pulmonary type A Francisella tularensis infection

Pneumonic tularemia is a potentially fatal disease caused by the Category A bioterrorism agent Francisella tularensis. Understanding the pulmonary immune response to this bacterium is necessary for developing effective vaccines and therapeutics. In this study, characterization of immune cell populations in the lungs of mice infected with the type A strain Schu S4 revealed a significant loss in natural killer (NK) cells over time. Since this decline in NK cells correlated with morbidity and mortality, we hypothesized these cells contribute to host defense against Schu S4 infection. Depletion of NK cells prior to Schu S4 challenge significantly reduced IFN-γ and granzyme B in the lung but had no effect on bacterial burden or disease progression. Conversely, increasing NK cell numbers with the anti-apoptotic cytokine IL-15 and soluble receptor IL-15Rα had no significant impact on Schu S4 growth in vivo. A modest decrease in median time to death, however, was observed in live vaccine strain (LVS)-vaccinated mice depleted of NK1.1⁺ cells and challenged with Schu S4. Therefore, NK cells do not appear to contribute to host defense against acute respiratory infection with type A F. tularensis in vivo, but they play a minor role in protection elicited by LVS vaccination.     

Persistence of Francisella tularensis McCoy et Chapin tularemia agent in the organism of highly sensitive rodents after oral infection

In the literature there are no data on the possibility of obtaining in experiment non-fatal tularemia infection (persistence) in rodents highly sensitive to it (Group I) when using highly virulent strains circulating in nature for infection by natural routes. Our detailed experiments on 1483 adult voles Microtus rossiaemeridionalis Ogn. (syn. M. subarvalis Meyer et al.) of laboratory origin using virulent strains of Francisella tularensis holarctica Ols. et Meshch. and natural alimentary infection by feeding on bodies of died animals or forced dosed administration of a mixture of dead and living bacteria to the voles through the oesophagus demonstrated the possibility of the animals to survive tularemia with subsequent long-term chronic carrier state of the infectious agent. They also confirmed the ability of voles to eat readily cadavers of their kin (cannibalism, necrophagia). Experiments with the fully virulent strain 503 and feeding on cadavers were carried out on 439 voles. 203 animals died from acute tularemia, 43 from side effects and 193 survived. Two of the latter (0.5%) exhibited chronic bacterial carrier state, and agglutinins to tularemia microbe (1:320) were found in their blood. From 309 voles subjected to dosed feeding, 153 died from acute tularemia, 27 from side effects and 129 survived. Two of them were bacterial carriers and 6 (1.9%) showed agglutinins (1:160-1:1280). In experiments with strain 165, spontaneously less virulent for guinea pigs, 433 voles were fed on cadavers. 170 of them died from acute tularemia, 53 from side effects, and 210 animals survived. Among the latter, 14 animals (3.2%) were found immune to 100 LD50 of the highly virulent strain 1298. In dosed feeding of 302 voles with the strain 165, 90 animals died from acute tularemia, 59 from side effects, and 153 survived, including 63 animals (20.7%) immune to 100 LD50. The surviving immune voles exhibited seroconversion and long-term persistence of the infectious agent in the internal organs (up to day 257-313--period of observation), accompanied bacteriuria in some cases. Histological examination of the kidney revealed, for the first time, important pathological changes of glomerulonephritis type with elements of pyelonephritis. Protracted stay of the agent in the organism of the vole does not affect its virulence. Persistence of tularemia agent in the organism of voles highly sensitive to tularemia in alimentary administration to them of living and dead bacteria is achieved as a result of anticipatory development of immunological reactions in response to a massive dose of killed antigen, against the background of which the accumulation of simultaneously administered     

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.     

Comparative proteome analysis of cellular proteins extracted from highly virulent Francisella tularensis ssp. tularensis and less virulent F. tularensis ssp. holarctica and F. tularensis ssp. mediaasiatica

Francisella tularensis is the causative agent of the zoonotic disease tularemia. Four subspecies of this pathogen, namely ssp. tularensis, mediaasiatica, holarctica, and novicida are spread throughout the northern hemisphere. Although there are marked variations in their virulence to mammals, the subspecies are difficult to identify as they are closely genetically related. We carried out the comparative proteome analysis of cellular extracts from isolates representing the highly virulent subspecies tularensis, and the less virulent subspecies mediaasiatica and holarctica in order to identify new diagnostic markers and putative factors of virulence. We identified 27 protein spots that were either specifically present or at significantly higher abundance in ssp. tularensis strains, 22 proteins in ssp. mediaasiatica strains, and 26 proteins in ssp. holarctica strains. Subspecies tularensis-specific proteins might represent putative virulence factors. Of 27 identified tularensis-specific spots 17 represented charge and mass variants of proteins occurring in other subspecies, 7 spots were found to be present at higher abundance, and 3 spots were specifically present in tularensis strains. Amongst them, PilP protein, as a component necessary for the biogenesis of the type IV pilus, virulence and adhesion factor for many human pathogen, was identified. Furthermore, the identification of additional 27 proteins common for ssp. tularensis and mediaasiatica, and 19 proteins shared by ssp. mediaasiatica and holarctica documented apparent closer genetic similarity between ssp. tularensis and mediaasiatica.     

Cytokine expression in the liver of mice infected with a highly virulent strain of Francisella tularensis

Abstract Cytokine mRNA expression was determined in the liver of mice subcutaneously inoculated with a lethal dose of the highly virulent strain FSC 041 of Francisella tularensis subvar. tularensis or a sublethal dose of the live vaccine strain of F. tularensis subvar. palaearctica . Expression of mRNA for TNF-α, IL-12, IFN-γ, and IL-10 was demonstrated within 48 h of inoculation, the kinetics being similar irrespective of bacterial strain used. Thus, the expression of a cytokine response believed to be important in the early host defence against live vaccine strain seemed insufficient to prevent the lethality of a more virulent strain.     

Evaluation of Francisella tularensis ΔpdpC as a candidate live attenuated vaccine against respiratory challenge by a virulent SCHU P9 strain of Francisella tularensis in a C57BL/6J mouse model

Francisella tularensis, which causes tularemia, is an intracellular gram‐negative bacterium. F. tularensis has received significant attention in recent decades because of its history as a biological weapon. Thus, development of novel vaccines against tularemia has been an important goal. The attenuated F. tularensis strain ΔpdpC, in which the pathogenicity determinant protein C gene (pdpC) has been disrupted by TargeTron mutagenesis, was investigated as a potential vaccine candidate for tularemia in the present study. C57BL/6J mice immunized s.c. with 1 × 10⁶ CFUs of ΔpdpC were challenged intranasally with 100× the median lethal dose (LD₅₀) of a virulent SCHU P9 strain 21 days post immunization. Protection against this challenge was achieved in 38% of immunized C57BL/6J mice administered 100 LD₅₀ of this strain. Conversely, all unimmunized mice succumbed to death 6 days post challenge. Survival rates were significantly higher in vaccinated than in unimmunized mice. In addition, ΔpdpC was passaged serially in mice to confirm its stable attenuation. Low bacterial loads persisted in mouse spleens during the first to tenth passages. No statistically significant changes in the number of CFUs were observed during in vivo passage of ΔpdpC. The inserted intron sequences for disrupting pdpC were completely maintained even after the tenth passage in mice. Considering the stable attenuation and intron sequences, it is suggested that ΔpdpC is a promising tularemia vaccine candidate.     

Tick salivary gland extract accelerates proliferation of Francisella tularensis in the host

Accelerated proliferation of the tick-borne bacterial pathogen Francisella tularensis was demonstrated in mice when the bacterium was injected together with salivary gland extract from Ixodes ricinus ticks. A significant increase in the numbers of bacteria was recorded in the dermal site of infection,the draining lymph nodes, and the spleen. Analysis of the expression of cytokine messenger ribonucleic acids showed polarization toward a Th2 profile. Salivary gland extract-mediated suppression of interleukin-12 and interferon-gamma, the cytokines required for the expression of the protective immunity against tularemic infection, apparently contributed to the decreased resistance against this tick-transmitted pathogen.     

Francisella tularensis live vaccine strain induces macrophage alternative activation as a survival mechanism

Francisella tularensis (Ft), the causative agent of tularemia, elicits a potent inflammatory response early in infection, yet persists within host macrophages and can be lethal if left unchecked. We report in this study that Ft live vaccine strain (LVS) infection of murine macrophages induced TLR2-dependent expression of alternative activation markers that followed the appearance of classically activated markers. Intraperitoneal infection with Ft LVS also resulted in induction of alternatively activated macrophages (AA-Mphi). Induction of AA-Mphi by treatment of cells with rIL-4 or by infection with Ft LVS promoted replication of intracellular Ftn, in contrast to classically activated (IFN-gamma plus LPS) macrophages that promoted intracellular killing of Ft LVS. Ft LVS failed to induce alternative activation in IL-4Ralpha(-/-) or STAT6(-/-) macrophages and prolonged the classical inflammatory response in these cells, resulting in intracellular killing of Ft. Treatment of macrophages with anti-IL-4 and anti-IL-13 Ab blunted Ft-induced AA-Mphi differentiation and resulted in increased expression of IL-12 p70 and decreased bacterial replication. In vivo, Ft-infected IL-4Ralpha(-/-) mice exhibited increased survival compared with wild-type mice. Thus, redirection of macrophage differentiation by Ft LVS from a classical to an alternative activation state enables the organism to survive at the expense of the host.     

Genomic comparison between a virulent type A1 strain of Francisella tularensis and its attenuated O-antigen mutant

We report the complete genome sequences of TI0902, a highly virulent type A1 strain, and TIGB03, a related, attenuated chemical mutant strain. Compared to the wild type, the mutant strain had 45 point mutations and a 75.9-kb duplicated region that had not been previously observed in Francisella species.     

Comparative proteomic profiling of culture filtrate proteins of less and highly virulent Francisella tularensis strains

The facultative intracellular bacterium Francisella tularensis is the causal agent of the serious infectious disease tularemia. Despite the dynamic progress, which has been made in last few years, important questions regarding Francisella pathogenicity still remain to be answered. Generally, secreted proteins play an important role in pathogenicity of intracellular microbes. In this study, we investigated the protein composition of the culture filtrate proteins of highly virulent F. tularensis subsp. tularensis, strain SCHU S4 and attenuated F. tularensis subsp. holarctica, live vaccine strain using a comparative proteomic analysis. The majority of proteins identified in this study have been implicated in virulence mechanisms of other pathogens, and several have been categorized as having moonlighting properties; those that have more than one unrelated function. This profiling study of secreted proteins resulted in the unique detection of acid phosphatase (precursor) A (AcpA), β-lactamase, and hypothetical protein FTT0484 in the highly virulent strain SCHU S4 secretome. The release of AcpA may be of importance for F. tularensis subsp. tularensis virulence due to the recently described AcpA role in the F. tularensis escape from phagosomes.     

Nucleolin, a shuttle protein promoting infection of human monocytes by Francisella tularensis

Background

Francisella tularensis is a highly virulent facultative intracellular bacterium, disseminating in vivo mainly within host mononuclear phagocytes. After entry into macrophages, F. tularensis initially resides in a phagosomal compartment, whose maturation is then arrested. Bacteria escape rapidly into the cytoplasm, where they replicate freely. We recently demonstrated that nucleolin, an eukaryotic protein able to traffic from the nucleus to the cell surface, acted as a surface receptor for F. tularensis LVS on human monocyte-like THP-1 cells.

Methodology/Principal Findings

Here, we followed the fate of nucleolin once F. tularensis has been endocytosed. We first confirmed by siRNA silencing experiments that expression of nucleolin protein was essential for binding of LVS on human macrophage-type THP-1 cells. We then showed that nucleolin co-localized with intracellular bacteria in the phagosomal compartment. Strikingly, in that compartment, nucleolin also co-localized with LAMP-1, a late endosomal marker. Co-immunoprecipation assays further demonstrated an interaction of nucleolin with LAMP-1. Co-localization of nucleolin with LVS was no longer detectable at 24 h when bacteria were multiplying in the cytoplasm. In contrast, with an iglC mutant of LVS, which remains trapped into the phagosomal compartment, or with inert particles, nucleolin/bacteria co-localization remained almost constant.

Conclusions/Significance

We herein confirm the importance of nucleolin expression for LVS binding and its specificity as nucleolin is not involved in binding of another intracellular pathogen as L. monocytogenes or an inert particle. Association of nucleolin with F. tularensis during infection continues intracellularly after endocytosis of the bacteria. The present work therefore unravels for the first time the presence of nucleolin in the phagosomal compartment of macrophages.     

Construction of a shuttle vector for use in Francisella tularensis

Abstract The characterisation of virulence factors of Francisella tularensis has been hampered by the lack of genetic system for the bacterium. In this study, a shuttle vector was constructed that can replicate autonomously in F. tularensis and Escherichia coli . To obtain this vector, the p15A replication origin of E. coli plasmid pACYC184 was introduced into a plasmid derivative of plasmid pFNL200, a plasmid which only can replicate in F. tularensis . The resulting shuttle vector, designated pKK202, harboured resistance genes for chloramphenicol and tetracycline. This vector might be used as a basis for the studies of virulence factors of F. tularensis .     

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.