iTRAQ quantitative analysis of Francisella tularensis ssp. holarctica live vaccine strain and Francisella tularensis ssp. tularensis SCHU S4 response to different temperatures and stationary phases of growth

Proteomics has been shown to significantly contribute to the investigation of the pathogenicity of the extremely infectious bacteria Francisella tularensis. In this study, the authors employed iTRAQ quantitative proteomic analysis in order to monitor alterations in proteomes of F. tularensis ssp. holarctica live vaccine strain and F. tularensis ssp. tularensis SCHU S4 associated with the cultivation at different temperatures or in the stationary phase. Correlated production of the identified proteins studied by the exploratory statistical analysis revealed novel candidates for virulence factors that were regulated in a similar manner to the genes encoded in the Francisella Pathogenicity Island. Moreover, the assessment of the adaptation of live vaccine strain and SCHU S4 strain to the examined stimuli uncovered differences in their physiological responses to the stationary phase of growth.     

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.     

Virulence of representative Japanese Francisella tularensis and immunologic consequences of infection in mice

Francisella tularensis, which causes tularemia, is widely distributed in the Northern hemisphere. F. tularensis strains isolated in Japan are genetically unique from non‐Japanese strains; however, their phenotypic properties have not been well studied. Thus, mice were infected with representative Japanese strains of F. tularensis and their virulence and mouse immune responses to them assessed. Of four representative Japanese strains, the Ebina, Jap and Tsuchiya strains were susceptible to H₂O₂ and did not grow well intracellularly. Only Yama strain grew intracellularly and was lethal to mice. Infection with Yama strain resulted in drastic increases in IFN‐γ, CD4 and CD8 double‐positive T cells and Th1 cells (CD3, CD4 and Tim3‐positive cells), and a decrease in the ratio of CD8‐positive CD4‐negative T cells in mice. C57BL/6J mice that survived infection produced IgM antibodies to LPS and IgG2c antibodies to 43, 19 and 17 kDa proteinase K‐sensitive components. These data are valuable for understanding the phenotypic properties of F. tularensis in Japan.     

The AcrAB RND efflux system from the live vaccine strain of Francisella tularensis is a multiple drug efflux system that is required for virulence in mice

The ability of bacterial pathogens to infect and cause disease is dependent upon their ability to resist antimicrobial components produced by their host, such as bile acids, fatty acids and other detergent-like molecules, and products of the innate immune system (e.g. cationic antimicrobial peptides). Bacterial resistance to the antimicrobial effects of such compounds is often mediated by active efflux systems belonging to the resistance-nodulation-division (RND) family of transporters. RND efflux systems have been implicated in antibiotic resistance and virulence extending their clinical relevance. In this report the hypothesis that the Francisella tularensis AcrAB RND efflux system contributes to antimicrobial resistance and pathogenesis has been tested. A null mutation was generated in the gene encoding the AcrB RND efflux pump protein of the live vaccine strain of F. tularensis. The resulting mutant exhibited increased sensitivity to multiple antibiotics and antimicrobial compounds. Murine challenge experiments revealed that the acrB mutant was attenuated. Collectively these results suggest that the F. tularensis AcrAB RND efflux system encodes a multiple drug efflux system that is important for virulence.     

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.     

Insights into the oxidative stress response in Francisella tularensis LVS and its mutant DeltaiglC1+2 by proteomics analysis

Francisella tularensis is a facultative intracellular pathogen. Its capacity to induce disease depends on the ability to invade and multiply within a wide range of eukaryotic cells, such as professional phagocytes. The comparative disinterest in tularemia in the past relative to other human bacterial pathogens is reflected in the paucity of information concerning the mechanisms of pathogenesis. Only a few genes and gene products associated with Francisella virulence are known to date. The aim of this study was to find and identify proteins of F. tularensis live vaccine strain induced in the presence of hydrogen peroxide, and to investigate the role of the IglC protein in the regulation of genes expressed upon peroxide stress. The [(35)S]-radiolabelled protein patterns were examined for both the wild live vaccine strain and its DeltaiglC1+2 mutant defective in synthesis of the IglC protein that was found to be strongly up-regulated during intracellular growth in murine macrophages in vitro and upon exposure to hydrogen peroxide. Globally, we found 21 protein spots whose levels were significantly altered in the presence of hydrogen peroxide in both the wild-type and mutant strains.     

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.     

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.     

Structure and Function of REP34 Implicates Carboxypeptidase Activity in Francisella tularensis Host Cell Invasion

Francisella tularensis is the etiological agent of tularemia, or rabbit fever. Although F. tularensis is a recognized biothreat agent with broad and expanding geographical range, its mechanism of infection and environmental persistence remain poorly understood. Previously, we identified seven F. tularensis proteins that induce a rapid encystment phenotype (REP) in the free-living amoeba, Acanthamoeba castellanii. Encystment is essential to the pathogen''s long term intracellular survival in the amoeba. Here, we characterize the cellular and molecular function of REP34, a REP protein with a mass of 34 kDa. A REP34 knock-out strain of F. tularensis has a reduced ability to both induce encystment in A. castellanii and invade human macrophages. We determined the crystal structure of REP34 to 2.05-Å resolution and demonstrate robust carboxypeptidase B-like activity for the enzyme. REP34 is a zinc-containing monomeric protein with close structural homology to the metallocarboxypeptidase family of peptidases. REP34 possesses a novel topology and substrate binding pocket that deviates from the canonical funnelin structure of carboxypeptidases, putatively resulting in a catalytic role for a conserved tyrosine and distinct S1′ recognition site. Taken together, these results identify REP34 as an active carboxypeptidase, implicate the enzyme as a potential key F. tularensis effector protein, and may help elucidate a mechanistic understanding of F. tularensis infection of phagocytic cells.     

Immunogenicity of a new lot of Francisella tularensis live vaccine strain in human volunteers

Abstract A new lot of Francisella tularensis live vaccine strain (LVS) was tested for immunogenicity in 19 human volunteers. Scarification vaccination induced specific cell-mediated and humoral immune responses. We noted a significant rise in antibodies against irradiation-killed LVS, formalin-killed virulent strain SCHU4, and an ether extracted antigen preparation (EEx) beginning 14 days after vaccination. A main target of the humoral immune response was lipopolysaccharide. Eighty percent of vaccinated volunteers developed a positive IgG response to EEx by day 14 and 100% of vaccinees responded positively by day 21. Background IgA titers were lower than corresponding IgG or IgM titers. No early IgM rise was noted with any antigen. By day 14 after vaccination, in vitro lymphocyte responses to LVS, the rough variant of LVS, and EEx were significantly increased compared to controls. Seventy percent of volunteers had a positive in vitro lymphocyte response to EEx within 14 days of vaccination. We predict that EEx will be a usefull antigen for diagnosing tularemia and for evaluating the immunogenicity of vaccines against tularemia. We are testing this antigen using sera from human cases of tularemia and control sera.     

Paralogous outer membrane proteins mediate uptake of different forms of iron and synergistically govern virulence in Francisella tularensis tularensis

Francisella tularensis subsp. tularensis is a highly infectious bacterium causing acute disease in mammalian hosts. Mechanisms for the acquisition of iron within the iron-limiting host environment are likely to be critical for survival of this intracellular pathogen. FslE (FTT0025) and FupA (FTT0918) are paralogous proteins that are predicted to form β-barrels in the outer membrane of virulent strain Schu S4 and are unique to Francisella species. Previous studies have implicated both FupA, initially identified as a virulence factor and FslE, encoded by the siderophore biosynthetic operon, in iron acquisition. Using single and double mutants, we demonstrated that these paralogs function in concert to promote growth under iron limitation. We used a (55)Fe transport assay to demonstrate that FslE is involved in siderophore-mediated ferric iron uptake, whereas FupA facilitates high affinity ferrous iron uptake. Optimal replication within J774A.1 macrophage-like cells required at least one of these uptake systems to be functional. In a mouse model of tularemia, the ΔfupA mutant was attenuated, but the ΔfslE ΔfupA mutant was significantly more attenuated, implying that the two systems of iron acquisition function synergistically to promote virulence. These studies highlight the importance of specific iron acquisition functions, particularly that of ferrous iron, for virulence of F. tularensis in the mammalian host.     

3-Deoxy-d-manno-octulosonic Acid (Kdo) Hydrolase Identified in Francisella tularensis,Helicobacter pylori,and Legionella pneumophila

3-Deoxy-d-manno-octulosonic acid (Kdo) is an eight-carbon sugar ubiquitous in Gram-negative bacterial lipopolysaccharides (LPS). Although its biosynthesis is well described, no protein has yet been identified as a Kdo hydrolase. However, Kdo hydrolase enzymatic activity has been detected in membranes of Helicobacter pylori and Francisella tularensis and may be responsible for the removal of side-chain Kdo from the LPS core saccharides. We now report the identification of genes encoding a Kdo hydrolase in F. tularensis Schu S4 and live vaccine strain strains, in H. pylori 26695 strain and in Legionella pneumophila Philadelphia 1 strain. We have renamed the genes kdhA for keto-deoxyoctulosonate hydrolase A. Deletion of kdhA abolished Kdo hydrolase activity in membranes of F. tularensis live vaccine strain. The F. tularensis kdhA mutant synthesized a core oligosaccharide containing a Kdo disaccharide with one of the Kdo residues being a terminal side chain. This side-chain Kdo monosaccharide was absent in the wild-type core oligosaccharide. Expression in Escherichia coli of recombinant KdhA from F. tularensis, H. pylori, and L. pneumophila resulted in a reduction of membrane-associated side-chain Kdo. The identification of this previously faceless enzyme will accelerate study of the biosynthetic basis and biologic impact for postbiosynthetic LPS structural modification.     

Identification of a novel Francisella tularensis factor required for intramacrophage survival and subversion of innate immune response

Francisella tularensis, the causative agent of tularemia, is one of the deadliest agents of biological warfare and bioterrorism. Extremely high virulence of this bacterium is associated with its ability to dampen or subvert host innate immune response. The objectives of this study were to identify factors and understand the mechanisms of host innate immune evasion by F. tularensis. We identified and explored the pathogenic role of a mutant interrupted at gene locus FTL_0325, which encodes an OmpA-like protein. Our results establish a pathogenic role of FTL_0325 and its ortholog FTT0831c in the virulent F. tularensis SchuS4 strain in intramacrophage survival and suppression of proinflammatory cytokine responses. This study provides mechanistic evidence that the suppressive effects on innate immune responses are due specifically to these proteins and that FTL_0325 and FTT0831c mediate immune subversion by interfering with NF-κB signaling. Furthermore, FTT0831c inhibits NF-κB activity primarily by preventing the nuclear translocation of p65 subunit. Collectively, this study reports a novel F. tularensis factor that is required for innate immune subversion caused by this deadly bacterium.     

Molecular immune responses to aerosol challenge with Francisella tularensis in mice inoculated with live vaccine candidates of varying efficacy

Background

Francisella tularensis is a facultative intracellular bacterial pathogen and the etiological agent of tularemia. The subspecies F. tularensis tularensis is especially virulent for humans when inhaled and respiratory tularemia is associated with high mortality if not promptly treated. A live vaccine strain (LVS) derived from the less virulent holarctica subspecies confers incomplete protection against aerosol challenge with subsp. tularensis. Moreover, correlates of protection have not been established for LVS.

Methodology/Principal Findings

In the present study we compare molecular immune responses elicited by LVS and two defined deletion mutants of clinical subsp. tularensis strain, SCHU S4, that confer enhanced protection in a mouse model. BALB/c mice were immunized intradermally then challenged with an aerosol of SCHU S4 six weeks later. Changes in the levels of a selected panel of cytokines and chemokines were examined in the lungs, spleens, and sera of vaccinated and challenged mice. Mostly, increased cytokine and chemokine levels correlated with increased bacterial burden. However, after adjusting for this variable, immunization with either of the two Schu S4 mutants resulted in higher levels of several pulmonary cytokines, versus those resulting after LVS immunization, including IL-17. Moreover, treatment of mice immunized with ΔclpB with anti-IL-17 antibodies post-challenge enhanced lung infection.

Conclusions/Significance

This is the first report characterizing local and systemic cytokine and chemokine responses in mice immunized with vaccines with different efficacies against aerosol challenge with virulent F. tularensis subsp. tularensis. It shows that increases in the levels of most of these immunomodulators, including those known to be critical for protective immunity, do not superficially correlate with protection unless adjusted for the effects of bacterial burden. Additionally, several cytokines were selectively suppressed in the lungs of naïve mice, suggesting that one mechanism of vaccine action is to overcome this pathogen-induced immunosuppression.     

Specificity of rabbit antisera against the rough lipopolysaccharide of Salmonella minnesota strain R7 (chemotype Rd1P−)

Abstract Rabbit polyclonal antibodies against the lipopolysaccharide (LPS) of the Rd₁P⁻ mutant strain R7 of Salmonella minnesota were serologically characterized using R7 LPS, dephosphorylated LPS, deacylated LPS, deacylated, dephosphorylated and reduced LPS, and synthetic partial structures. The latter comprised partial structures of the core region of Rd₁P⁻ LPS bound to the β 1 → 6-linked glucosamine disaccharide with two amide-linked 3-hydroxytetradecanoic acid residues or artificial glycoconjugates comprised of the synthetic oligosaccharides coupled to bovine serum albumin. Using a passive hemolysis and an enzyme immunoassay, absorption and inhibition experiments, the antibody specificities present could be determined. One group of antibodies required components of the core region and the phosphorylated glucosamine disaccharide of the lipid A moiety for binding. The majority of phosphate-independent antibodies was directed against the trisaccharide l -glycero-α- d -manno-heptopyranose(1 → 3)- l -glycero-α- d -manno-heptopyranose(1 → 5)3-deoxy- d -manno-octulosonic acid. Antibodies against the 1 → 3- and 1 → 7-linked heptose disaccharides and against a single heptose were also detected, however, with low titers. No antibodies were found which required the presence of fatty acids.     

The Entry and Intracellular Multiplication of Francisella tularensis in Cultured Cells: Its Correlation with Virulence in Experimental Mice

Five acriflavine agglutination test-positive (acf+) colonies and five negative (acf–) colonies were isolated from each of the four strains (Ebina, CMB2, N9, and Schu) of Francisella tularensis, and the correlation between the virulence in experimental mice and the entry and intracellular multiplication in cultured mouse fibroblast cells (L-929 cells) was examined. All of the acf– colonies derived from the Ebina and CMB2 strains were highly virulent in mice, readily entering and growing well in the cells, while all of the acf- colonies from N9 and Schu strains were of low virulence and neither entered nor grew in the cells effectively. On the other hand, regardless of their parent strains, the acf+ colonies were low virulent and most of those colonies did neither enter nor grow in L-929 cells. In addition, two acf- colonies, one from the N9 and the other from the Schu strain, gained virulence through several passages in mice, and in parallel, their entry and multiplication also improved. However, two acf+ colonies from the Ebina strain and one acf+ colony from the N9 strain showed a moderate degree of the entry and multiplication although they were all low virulent. The overall results indicate that the entry and multiplication in cells are important factors regulating the virulence of F. tularensis. The results also showed, however, that they were not sole factors to elucidate the virulence of the bacterium in mice.     

Isolation of a Francisella tularensis mutant that is sensitive to serum and oxidative killing and is avirulent in mice: Correlation with the loss of MinD homologue expression

Abstract We constructed mutant strains of Francisella tularensis biotype novicida by insertional mutagenesis with a kanamycin resistance (Km^R) cassette. One mutant, KEM7, was defective for survival in macrophages in comparison with the wild-type (WT) strain and a random insertion strain, KEM21. While all three strains exhibited intracellular growth, the number of viable KEM7 present after 24–48 h of infection was approximately 10 times less than that of WT or KEM21. This observation was apparently due to a reduced number of viable KEM7 associated with the macrophages one hour after phagocytosis. KEM7 was approximately 3 times more susceptible than WT or KEM21 to killing by the products of the xanthine-xanthine oxidase reaction or by hydrogen peroxide. KEM7 was also found to be susceptible to killing by serum, whereas WT and KEM21 were resistant. Upon intravenous inoculation of C57BL/6 mice, the number of KEM7 in the livers and spleens 48 h post-infection was found to be 1000- to 10 000-times less than that of either KEM21 or WT. DNA sequence analysis at the Km^R insertion site suggested that the F. tularensis homologue of min D had been interrupted. Western immunoblot analysis confirmed the presence of a MinD homologue in F. tularensis WT and KEM21, and demonstrated its absence in KEM7.