Type A Francisella tularensis Acid Phosphatases Contribute to Pathogenesis

Different Francisella spp. produce five or six predicted acid phosphatases (AcpA, AcpB, AcpC, AcpD, HapA and HapB). The genes encoding the histidine acid phosphatases (hapA, hapB) and acpD of F. tularensis subsp. Schu S4 strain are truncated or disrupted. However, deletion of HapA (FTT1064) in F. tularensis Schu S4 resulted in a 33% reduction in acid phosphatase activity and loss of the four functional acid phosphatases in F. tularensis Schu S4 (ΔABCH) resulted in a>99% reduction in acid phosphatase activity compared to the wild type strain. All single, double and triple mutants tested, demonstrated a moderate decrease in mouse virulence and survival and growth within human and murine phagocytes, whereas the ΔABCH mutant showed >3.5-fold decrease in intramacrophage survival and 100% attenuation of virulence in mouse. While the Schu S4 ΔABCH strain was attenuated in the mouse model, it showed only limited protection against wild type challenge. F. tularensis Schu S4 failed to stimulate reactive oxygen species production in phagocytes, whereas infection by the ΔABCH strain stimulated 5- and 56-fold increase in reactive oxygen species production in neutrophils and human monocyte-derived macrophages, respectively. The ΔABCH mutant but not the wild type strain strongly co-localized with p47^phox and replicated in macrophages isolated from p47^phox knockout mice. Thus, F. tularensis Schu S4 acid phosphatases, including the truncated HapA, play a major role in intramacrophage survival and virulence of this human pathogen.     

Interactions of Francisella tularensis with Alveolar Type II Epithelial Cells and the Murine Respiratory Epithelium

Francisella tularensis is classified as a Tier 1 select agent by the CDC due to its low infectious dose and the possibility that the organism can be used as a bioweapon. The low dose of infection suggests that Francisella is unusually efficient at evading host defenses. Although ~50 cfu are necessary to cause human respiratory infection, the early interactions of virulent Francisella with the lung environment are not well understood. To provide additional insights into these interactions during early Francisella infection of mice, we performed TEM analysis on mouse lungs infected with F. tularensis strains Schu S4, LVS and the O-antigen mutant Schu S4 waaY::TrgTn. For all three strains, the majority of the bacteria that we could detect were observed within alveolar type II epithelial cells at 16 hours post infection. Although there were no detectable differences in the amount of bacteria within an infected cell between the three strains, there was a significant increase in the amount of cellular debris observed in the air spaces of the lungs in the Schu S4 waaY::TrgTn mutant compared to either the Schu S4 or LVS strain. We also studied the interactions of Francisella strains with human AT-II cells in vitro by characterizing the ability of these three strains to invade and replicate within these cells. Gentamicin assay and confocal microscopy both confirmed that F. tularensis Schu S4 replicated robustly within these cells while F. tularensis LVS displayed significantly lower levels of growth over 24 hours, although the strain was able to enter these cells at about the same level as Schu S4 (1 organism per cell), as determined by confocal imaging. The Schu S4 waaY::TrgTn mutant that we have previously described as attenuated for growth in macrophages and mouse virulence displayed interesting properties as well. This mutant induced significant airway inflammation (cell debris) and had an attenuated growth phenotype in the human AT-II cells. These data extend our understanding of early Francisella infection by demonstrating that Francisella enter significant numbers of AT-II cells within the lung and that the capsule and LPS of wild type Schu S4 helps prevent murine lung damage during infection. Furthermore, our data identified that human AT-II cells allow growth of Schu S4, but these same cells supported poor growth of the attenuated LVS strain in vitro. Collectively, these data further our understanding of the role of AT-II cells in Francisella infections.     

Fine Tuning Inflammation at the Front Door: Macrophage Complement Receptor 3-mediates Phagocytosis and Immune Suppression for Francisella tularensis

Complement receptor 3 (CR3, CD11b/CD18) is a major macrophage phagocytic receptor. The biochemical pathways through which CR3 regulates immunologic responses have not been fully characterized. Francisella tularensis is a remarkably infectious, facultative intracellular pathogen of macrophages that causes tularemia. Early evasion of the host immune response contributes to the virulence of F. tularensis and CR3 is an important receptor for its phagocytosis. Here we confirm that efficient attachment and uptake of the highly virulent Type A F. tularensis spp. tularensis strain Schu S4 by human monocyte-derived macrophages (hMDMs) requires complement C3 opsonization and CR3. However, despite a>40-fold increase in uptake following C3 opsonization, Schu S4 induces limited pro-inflammatory cytokine production compared with non-opsonized Schu S4 and the low virulent F. novicida. This suggests that engagement of CR3 by opsonized Schu S4 contributes specifically to the immune suppression during and shortly following phagocytosis which we demonstrate by CD11b siRNA knockdown in hMDMs. This immune suppression is concomitant with early inhibition of ERK1/2, p38 MAPK and NF-κB activation. Furthermore, TLR2 siRNA knockdown shows that pro-inflammatory cytokine production and MAPK activation in response to non-opsonized Schu S4 depends on TLR2 signaling providing evidence that CR3-TLR2 crosstalk mediates immune suppression for opsonized Schu S4. Deletion of the CD11b cytoplasmic tail reverses the CR3-mediated decrease in ERK and p38 activation during opsonized Schu-S4 infection. The CR3-mediated signaling pathway involved in this immune suppression includes Lyn kinase and Akt activation, and increased MKP-1, which limits TLR2-mediated pro-inflammatory responses. These data indicate that while the highly virulent F. tularensis uses CR3 for efficient uptake, optimal engagement of this receptor down-regulates TLR2-dependent pro-inflammatory responses by inhibiting MAPK activation through outside-in signaling. CR3-linked immune suppression is an important mechanism involved in the pathogenesis of F. tularensis infection.     

Characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agent bacterium, Francisella tularensis Schu S4 and the surrogate type B live vaccine strain (LVS)

Here, we constructed stable, constitutively expressed, chromosomal green (GFP) and red fluorescent (RFP) reporters in the genome of the surrogate strain, Francisella tularensis spp. holarctica LVS (herein LVS), and the select agent, F. tularensis Schu S4. A bioinformatic approach was used to identify constitutively expressed genes. Two promoter regions upstream of the FTT1794 and rpsF(FTT1062) genes were selected and fused with GFP and RFP reporter genes in pMP815, respectively. While the LVS strains with chromosomally integrated reporter fusions exhibited fluorescence, we were unable to deliver the same fusions into Schu S4. Neither a temperature-sensitive Francisella replicon nor a pBBR replicon in the modified pMP815 derivatives facilitated integration. However, a mini-Tn7 integration system was successful at integrating the reporter fusions into the Schu S4 genome. Finally, fluorescent F. tularensis LVS and a mutant lacking MglA were assessed for growth in monocyte-derived macrophages (MDMs). As expected, when compared to wild-type bacteria, replication of an mglA mutant was significantly diminished, and the overall level of fluorescence dramatically decreased with infection time. The utility of the fluorescent Schu S4 strain was also examined within infected MDMs treated with clarithromycin and enrofloxacin. Taken together, this study describes the development of an important reagent for F. tularensis research, especially since the likelihood of engineered antibiotic resistant strains will emerge with time. Such strains will be extremely useful in high-throughput screens for novel compounds that could interfere with critical virulence processes in this important bioweapons agent and during infection of alveolar macrophages.     

Contribution of Citrulline Ureidase to Francisella tularensis Strain Schu S4 Pathogenesis

The citrulline ureidase (CTU) activity has been shown to be associated with highly virulent Francisella tularensis strains, including Schu S4, while it is absent in avirulent or less virulent strains. A definitive role of the ctu gene in virulence and pathogenesis of F. tularensis Schu S4 has not been assessed; thus, an understanding of the significance of this phenotype is long overdue. CTU is a carbon-nitrogen hydrolase encoded by the citrulline ureidase (ctu) gene (FTT0435) on the F. tularensis Schu S4 genome. In the present study, we evaluated the contribution of the ctu gene in the virulence of category A agent F. tularensis Schu S4 by generating a nonpolar deletion mutant, the Δctu mutant. The deletion of the ctu gene resulted in loss of CTU activity, which was restored by transcomplementing the ctu gene. The Δctu mutant did not exhibit any growth defect under acellular growth conditions; however, it was impaired for intramacrophage growth in resting as well as gamma interferon-stimulated macrophages. The Δctu mutant was further tested for its virulence attributes in a mouse model of respiratory tularemia. Mice infected intranasally with the Δctu mutant showed significantly reduced bacterial burden in the lungs, liver, and spleen compared to wild-type (WT) Schu S4-infected mice. The reduced bacterial burden in mice infected with the Δctu mutant was also associated with significantly lower histopathological scores in the lungs. Mice infected with the Δctu mutant succumbed to infection, but they survived longer and showed significantly extended median time to death compared to that shown by WT Schu S4-infected mice. To conclude, this study demonstrates that ctu contributes to intracellular survival, in vivo growth, and pathogenesis. However, ctu is not an absolute requirement for the virulence of F. tularensis Schu S4 in mice.Francisella tularensis, the etiological agent of tularemia, is a category A bioterrorism agent. High infectivity, ease of intentional aerosol dissemination, and lack of a licensed vaccine have made Francisella a potential biowarfare agent (5, 12, 34). The two major subspecies of Francisella have been divided on the basis of virulence, epidemiological distribution, and biochemical reactions (51). F. tularensis subspecies tularensis (type A strain) is highly virulent and the major cause of tularemia in North America, whereas F. tularensis subspecies holarctica (type B strain), prevalent in Europe and Asia, is less virulent. Biochemically, type A strains produce acid from glycerol and exhibit citrulline ureidase (CTU) activity, while type B strains do not exhibit these activities (21). In contrast to these biochemical differences, very limited variation is seen at the genetic level (25, 41), suggesting that differences in virulence between type A and B strains may arise from differential gene expression by nearly homologous genomes. The highly virulent Schu S4 strain represents type A F. tularensis subspecies tularensis and was originally isolated from a clinical case of tularemia in Ohio in 1941. To date, only a few virulence-associated genes have been characterized in this strain (22, 36, 37, 48), and its virulence determinants still remain poorly understood.CTU, a member of the carbon-nitrogen hydrolase family protein encoded by the F. tularensis genome (FTT0435), degrades citrulline into ornithine, carbon dioxide, and ammonia (10). Citrulline is generated during the catabolism of arginine by bacterial arginine deiminase (ADI) (40, 47). Ornithine generated by citrulline degradation is either exchanged for arginine by an arginine-ornithine transporter or utilized for the generation of polyamines and energy in the form of ATP (40). Citrulline is also produced by macrophages during conversion of l-arginine and oxygen to nitric oxide (NO) by inducible NO synthase (iNOS). Citrulline thus formed can be recycled to l-arginine through an arginine-citrulline cycle, which not only regulates intracellular availability of l-arginine but, in turn, maintains a sustained production of NO by macrophages (19). However, unlike citrulline, macrophages have little or no capacity to convert ornithine, the breakdown product of citrulline into l-arginine (4). Recent reports have demonstrated that reactive nitrogen species derived from NO are critical for clearance of F. tularensis (27, 29). In addition, ammonia generated by degradation of citrulline has been proposed to play a role in alkalization of endosomal pH leading to phagosomal maturation arrest (25). Thus, interruption of the arginine-citrulline cycle through the degradation of citrulline into ornithine, CO₂, and ammonia by CTU may assume an important role in the virulence of F. tularensis.Until recently, CTU activity has been used to differentiate strains of F. tularensis with high virulence from strains with low virulence or avirulent strains (45). Previous studies have shown that the majority of virulent F. tularensis type A strains exhibit high CTU activity while strains lacking this enzyme activity are either less virulent or avirulent (10, 11). However, a direct relationship between CTU activity and virulence of F. tularensis could not be established. A majority of these previous studies were based on comparisons of CTU activity in naturally occurring wild-type (WT) virulent type A strains with that in less virulent or avirulent type B variants of F. tularensis. In the current study, a genetic approach was used to directly assess the role of CTU activity in the pathogenesis and virulence of the F. tularensis Schu S4 strain.     

Comparative Genomic Characterization of Francisella tularensis Strains Belonging to Low and High Virulence Subspecies

Tularemia is a geographically widespread, severely debilitating, and occasionally lethal disease in humans. It is caused by infection by a gram-negative bacterium, Francisella tularensis. In order to better understand its potency as an etiological agent as well as its potential as a biological weapon, we have completed draft assemblies and report the first complete genomic characterization of five strains belonging to the following different Francisella subspecies (subsp.): the F. tularensis subsp. tularensis FSC033, F. tularensis subsp. holarctica FSC257 and FSC022, and F. tularensis subsp. novicida GA99-3548 and GA99-3549 strains. Here, we report the sequencing of these strains and comparative genomic analysis with recently available public Francisella sequences, including the rare F. tularensis subsp. mediasiatica FSC147 strain isolate from the Central Asian Region. We report evidence for the occurrence of large-scale rearrangement events in strains of the holarctica subspecies, supporting previous proposals that further phylogenetic subdivisions of the Type B clade are likely. We also find a significant enrichment of disrupted or absent ORFs proximal to predicted breakpoints in the FSC022 strain, including a genetic component of the Type I restriction-modification defense system. Many of the pseudogenes identified are also disrupted in the closely related rarely human pathogenic F. tularensis subsp. mediasiatica FSC147 strain, including modulator of drug activity B (mdaB) (FTT0961), which encodes a known NADPH quinone reductase involved in oxidative stress resistance. We have also identified genes exhibiting sequence similarity to effectors of the Type III (T3SS) and components of the Type IV secretion systems (T4SS). One of the genes, msrA2 (FTT1797c), is disrupted in F. tularensis subsp. mediasiatica and has recently been shown to mediate bacterial pathogen survival in host organisms. Our findings suggest that in addition to the duplication of the Francisella Pathogenicity Island, and acquisition of individual loci, adaptation by gene loss in the more recently emerged tularensis, holarctica, and mediasiatica subspecies occurred and was distinct from evolutionary events that differentiated these subspecies, and the novicida subspecies, from a common ancestor. Our findings are applicable to future studies focused on variations in Francisella subspecies pathogenesis, and of broader interest to studies of genomic pathoadaptation in bacteria.     

Control of Francisella tularensis Intracellular Growth by Pulmonary Epithelial Cells

The virulence of F. tularensis is often associated with its ability to grow in macrophages, although recent studies show that Francisella proliferates in multiple host cell types, including pulmonary epithelial cells. Thus far little is known about the requirements for killing of F. tularensis in the non-macrophage host cell types that support replication of this organism. Here we sought to address this question through the use of a murine lung epithelial cell line (TC-1 cells). Our data show that combinations of the cytokines IFN-γ, TNF, and IL-17A activated murine pulmonary epithelial cells to inhibit the intracellular growth of the F. tularensis Live Vaccine Strain (LVS) and the highly virulent F. tularensis Schu S4 strain. Although paired combinations of IFN-γ, TNF, and IL-17A all significantly controlled LVS growth, simultaneous treatment with all three cytokines had the greatest effect on LVS growth inhibition. In contrast, Schu S4 was more resistant to cytokine-induced growth effects, exhibiting significant growth inhibition only in response to all three cytokines. Since one of the main antimicrobial mechanisms of activated macrophages is the release of reactive nitrogen intermediates (RNI) via the activity of iNOS, we investigated the role of RNI and iNOS in Francisella growth control by pulmonary epithelial cells. NOS2 gene expression was significantly up-regulated in infected, cytokine-treated pulmonary epithelial cells in a manner that correlated with LVS and Schu S4 growth control. Treatment of LVS-infected cells with an iNOS inhibitor significantly reversed LVS killing in cytokine-treated cultures. Further, we found that mouse pulmonary epithelial cells produced iNOS during in vivo respiratory LVS infection. Overall, these data demonstrate that lung epithelial cells produce iNOS both in vitro and in vivo, and can inhibit Francisella intracellular growth via reactive nitrogen intermediates.     

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.     

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.     

Whole-Genome Sequencing Reveals Distinct Mutational Patterns in Closely Related Laboratory and Naturally Propagated Francisella tularensis Strains

The F. tularensis type A strain FSC198 from Slovakia and a second strain FSC043, which has attenuated virulence, are both considered to be derivatives of the North American F. tularensis type A strain SCHU S4. These strains have been propagated under different conditions: the FSC198 has undergone natural propagation in the environment, while the strain FSC043 has been cultivated on artificial media in laboratories. Here, we have compared the genome sequences of FSC198, FSC043, and SCHU S4 to explore the possibility that the contrasting propagation conditions may have resulted in different mutational patterns. We found four insertion/deletion events (INDELs) in the strain FSC043, as compared to the SCHU S4, while no single nucleotide polymorphisms (SNPs) or variable number of tandem repeats (VNTRs) were identified. This result contrasts with previously reported findings for the strain FSC198, where eight SNPs and three VNTR differences, but no INDELs exist as compared to the SCHU S4 strain. The mutations detected in the laboratory and naturally propagated type A strains, respectively, demonstrate distinct patterns supporting that analysis of mutational spectra might be a useful tool to reveal differences in past growth conditions. Such information may be useful to identify leads in a microbial forensic investigation.     

Clear Media for the Recovery of Pasteurella tularensis

Peptic digests of blood and hemoglobin were investigated as substitutes for the blood used in the preparation of glucose-cysteine-blood agar plating medium for the recovery of the virulent Schu strain of Pasteurella tularensis. Digest media so prepared were found to be satisfactory for the quantitative recovery of freshly grown cells but not for cells stored longer than several days. The addition of appropriate quantities of human plasma, bovine sera, or soluble starch rendered the digest media appropriate for use with stored cultures. The peptic digest-plasma (PDP) and peptic digest-starch (PDS) media were evaluated and found satisfactory for the quantitative recovery of P. tularensis Schu from freshly prepared and stored cultures, and from aerosols produced therefrom. With cultures stored longer than 6 weeks, the starch modification (PDS) was not as satisfactory as, and the plasma variation (PDP) was better than, glucose-cysteine-blood agar (GCBA) for the recovery of the organisms. PDP was superior to either GCBA or PDS medium for the recovery of the weakly virulent Jap 4 and Niieg-blue strains of P. tularensis.     

TaqMan Real-Time PCR Assays for Single-Nucleotide Polymorphisms Which Identify Francisella tularensis and Its Subspecies and Subpopulations

Francisella tularensis, the etiologic agent of tularemia and a Class A Select Agent, is divided into three subspecies and multiple subpopulations that differ in virulence and geographic distribution. Given these differences, there is a need to rapidly and accurately determine if a strain is F. tularensis and, if it is, assign it to subspecies and subpopulation. We designed TaqMan real-time PCR genotyping assays using eleven single nucleotide polymorphisms (SNPs) that were potentially specific to closely related groups within the genus Francisella, including numerous subpopulations within F. tularensis species. We performed extensive validation studies to test the specificity of these SNPs to particular populations by screening the assays across a set of 565 genetically and geographically diverse F. tularensis isolates and an additional 21 genetic near-neighbor (outgroup) isolates. All eleven assays correctly determined the genetic groups of all 565 F. tularensis isolates. One assay differentiates F. tularensis, F. novicida, and F. hispaniensis from the more genetically distant F. philomiragia and Francisella-like endosymbionts. Another assay differentiates F. tularensis isolates from near neighbors. The remaining nine assays classify F. tularensis-confirmed isolates into F. tularensis subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non F. tularensis), therefore a hierarchical approach of assay usage is recommended wherein the F. tularensis-specific assay is used before the nine downstream assays. Among F. tularensis isolates, all eleven assays were highly sensitive, consistently amplifying very low concentrations of DNA. Altogether, these eleven TaqMan real-time PCR assays represent a highly accurate, rapid, and sensitive means of identifying the species, subspecies, and subpopulation of any F. tularensis isolate if used in a step-wise hierarchical scheme. These assays would be very useful in clinical, epidemiological, and/or forensic investigations involving F. tularensis.     

Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains

Background

Francisella tularensis subspecies tularensis and holarctica are pathogenic to humans, whereas the two other subspecies, novicida and mediasiatica, rarely cause disease. To uncover the factors that allow subspecies tularensis and holarctica to be pathogenic to humans, we compared their genome sequences with the genome sequence of Francisella tularensis subspecies novicida U112, which is nonpathogenic to humans.

Results

Comparison of the genomes of human pathogenic Francisella strains with the genome of U112 identifies genes specific to the human pathogenic strains and reveals pseudogenes that previously were unidentified. In addition, this analysis provides a coarse chronology of the evolutionary events that took place during the emergence of the human pathogenic strains. Genomic rearrangements at the level of insertion sequences (IS elements), point mutations, and small indels took place in the human pathogenic strains during and after differentiation from the nonpathogenic strain, resulting in gene inactivation.

Conclusion

The chronology of events suggests a substantial role for genetic drift in the formation of pseudogenes in Francisella genomes. Mutations that occurred early in the evolution, however, might have been fixed in the population either because of evolutionary bottlenecks or because they were pathoadaptive (beneficial in the context of infection). Because the structure of Francisella genomes is similar to that of the genomes of other emerging or highly pathogenic bacteria, this evolutionary scenario may be shared by pathogens from other species.     

Identification of Genes Contributing to the Virulence of Francisella tularensis SCHU S4 in a Mouse Intradermal Infection Model

Background

Francisella tularensis is a highly virulent human pathogen. The most virulent strains belong to subspecies tularensis and these strains cause a sometimes fatal disease. Despite an intense recent research effort, there is very limited information available that explains the unique features of subspecies tularensis strains that distinguish them from other F. tularensis strains and that explain their high virulence. Here we report the use of targeted mutagenesis to investigate the roles of various genes or pathways for the virulence of strain SCHU S4, the type strain of subspecies tularensis.

Methodology/Principal Findings

The virulence of SCHU S4 mutants was assessed by following the outcome of infection after intradermal administration of graded doses of bacteria. By this route, the LD₅₀ of the SCHU S4 strain is one CFU. The virulence of 20 in-frame deletion mutants and 37 transposon mutants was assessed. A majority of the mutants did not show increased prolonged time to death, among them notably ΔpyrB and ΔrecA. Of the remaining, mutations in six unique targets, tolC, rep, FTT0609, FTT1149c, ahpC, and hfq resulted in significantly prolonged time to death and mutations in nine targets, rplA, wbtI, iglB, iglD, purL, purF, ggt, kdtA, and glpX, led to marked attenuation with an LD₅₀ of >10³ CFU. In fact, the latter seven mutants showed very marked attenuation with an LD₅₀ of ≥10⁷ CFU.

Conclusions/Significance

The results demonstrate that the characterization of targeted mutants yielded important information about essential virulence determinants that will help to identify the so far little understood extreme virulence of F. tularensis subspecies tularensis.     

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.     

Francisella tularensis regulates autophagy-related host cell signaling pathways

The Gram-negative intracellular pathogen Francisella tularensis is known for its ability to dampen host immune responses. We recently performed a microarray analsyis comparing human monocyte responses to the highly virulent F. tularensis tularensis Schu S4 strain (F.t.) versus the less virulent F. tularensis novicida (F.n.).¹ Many groups of genes were affected, including those involved with autophagy and with the regulation of autophagy. Here, we discuss the implications in the context of Francisella virulence and host cell response, then conclude with potential future experiments.     

Effect of a glucose impulse on the CcpA regulon in Staphylococcus aureus

Background

A low genetic diversity in Francisella tularensis has been documented. Current DNA based genotyping methods for typing F. tularensis offer a limited and varying degree of subspecies, clade and strain level discrimination power. Whole genome sequencing is the most accurate and reliable method to identify, type and determine phylogenetic relationships among strains of a species. However, lower cost typing schemes are necessary in order to enable typing of hundreds or even thousands of isolates.

Results

We have generated a high-resolution phylogenetic tree from 40 Francisella isolates, including 13 F. tularensis subspecies holarctica (type B) strains, 26 F. tularensis subsp. tularensis (type A) strains and a single F. novicida strain. The tree was generated from global multi-strain single nucleotide polymorphism (SNP) data collected using a set of six Affymetrix GeneChip^® resequencing arrays with the non-repetitive portion of LVS (type B) as the reference sequence complemented with unique sequences of SCHU S4 (type A). Global SNP based phylogenetic clustering was able to resolve all non-related strains. The phylogenetic tree was used to guide the selection of informative SNPs specific to major nodes in the tree for development of a genotyping assay for identification of F. tularensis subspecies and clades. We designed and validated an assay that uses these SNPs to accurately genotype 39 additional F. tularensis strains as type A (A1, A2, A1a or A1b) or type B (B1 or B2).

Conclusion

Whole-genome SNP based clustering was shown to accurately identify SNPs for differentiation of F. tularensis subspecies and clades, emphasizing the potential power and utility of this methodology for selecting SNPs for typing of F. tularensis to the strain level. Additionally, whole genome sequence based SNP information gained from a representative population of strains may be used to perform evolutionary or phylogenetic comparisons of strains, or selection of unique strains for whole-genome sequencing projects.     

Genomic Markers for Differentiation of Francisella tularensis subsp. tularensis A.I and A.II Strains

Tularemia is caused by two subspecies of Francisella tularensis, F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). F. tularensis subsp. tularensis is further subdivided into two genetically distinct populations (A.I and A.II) that differ with respect to geographical location, anatomical source of recovered isolates, and disease outcome. Using two human clinical isolates, suppression subtractive hybridization was performed to identify 13 genomic regions of difference between A.I and A.II strains. Two PCR assays, one to identify A.I and A.II as well as to discriminate between F. tularensis subsp. holarctica and F. novicida and another specific for A.I, were developed. This is the first report to identify and characterize conserved genomic differences between A.I and A.II.     

Complete genomic characterization of a pathogenic A.II strain of Francisella tularensis subspecies tularensis

Francisella tularensis is the causative agent of tularemia, which is a highly lethal disease from nature and potentially from a biological weapon. This species contains four recognized subspecies including the North American endemic F. tularensis subsp. tularensis (type A), whose genetic diversity is correlated with its geographic distribution including a major population subdivision referred to as A.I and A.II. The biological significance of the A.I - A.II genetic differentiation is unknown, though there are suggestive ecological and epidemiological correlations. In order to understand the differentiation at the genomic level, we have determined the complete sequence of an A.II strain (WY96-3418) and compared it to the genome of Schu S4 from the A.I population. We find that this A.II genome is 1,898,476 bp in size with 1,820 genes, 1,303 of which code for proteins. While extensive genomic variation exists between "WY96" and Schu S4, there is only one whole gene difference. This one gene difference is a hypothetical protein of unknown function. In contrast, there are numerous SNPs (3,367), small indels (1,015), IS element differences (7) and large chromosomal rearrangements (31), including both inversions and translocations. The rearrangement borders are frequently associated with IS elements, which would facilitate intragenomic recombination events. The pathogenicity island duplicated regions (DR1 and DR2) are essentially identical in WY96 but vary relative to Schu S4 at 60 nucleotide positions. Other potential virulence-associated genes (231) varied at 559 nucleotide positions, including 357 non-synonymous changes. Molecular clock estimates for the divergence time between A.I and A.II genomes for different chromosomal regions ranged from 866 to 2131 years before present. This paper is the first complete genomic characterization of a member of the A.II clade of Francisella tularensis subsp. tularensis.     

Kinetic Characterization and Phosphoregulation of the Francisella tularensis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

Deliberate and natural outbreaks of infectious disease underscore the necessity of effective vaccines and antimicrobial/antiviral therapeutics. The prevalence of antibiotic resistant strains and the ease by which antibiotic resistant bacteria can be intentionally engineered further highlights the need for continued development of novel antibiotics against new bacterial targets. Isoprenes are a class of molecules fundamentally involved in a variety of crucial biological functions. Mammalian cells utilize the mevalonic acid pathway for isoprene biosynthesis, whereas many bacteria utilize the methylerythritol phosphate (MEP) pathway, making the latter an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP synthase, a MEP pathway enzyme and potential target for antibiotic development. In vitro growth-inhibition assays using fosmidomycin, an inhibitor of MEP synthase, illustrates the effectiveness of MEP pathway inhibition with F. tularensis. To facilitate drug development, F. tularensis MEP synthase was cloned, expressed, purified, and characterized. Enzyme assays produced apparent kinetic constants (K_M^DXP = 104 µM, K_M^NADPH = 13 µM, k_cat^DXP = 2 s⁻¹, k_cat^NADPH = 1.3 s⁻¹), an IC₅₀ for fosmidomycin of 247 nM, and a K_i for fosmidomycin of 99 nM. The enzyme exhibits a preference for Mg⁺² as a divalent cation. Titanium dioxide chromatography-tandem mass spectrometry identified Ser177 as a site of phosphorylation. S177D and S177E site-directed mutants are inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP synthase is an excellent target for the development of novel antibiotics against F. tularensis.