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.     

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.     

Virulence Differences Among Francisella tularensis Subsp. tularensis Clades in Mice

Francisella tularensis subspecies tularensis (type A) and holarctica (type B) are of clinical importance in causing tularemia. Molecular typing methods have further separated type A strains into three genetically distinct clades, A1a, A1b and A2. Epidemiological analyses of human infections in the United States suggest that A1b infections are associated with a significantly higher mortality rate as compared to infections caused by A1a, A2 and type B. To determine if genetic differences as defined by molecular typing directly correlate with differences in virulence, A1a, A1b, A2 and type B strains were compared in C57BL/6 mice. Here we demonstrate significant differences between survival curves for infections caused by A1b versus A1a, A2 and type B, with A1b infected mice dying earlier than mice infected with A1a, A2 or type B; these results were conserved among multiple strains. Differences were also detected among type A clades as well as between type A clades and type B with respect to bacterial burdens, and gross anatomy in infected mice. Our results indicate that clades defined within F. tularensis subsp. tularensis by molecular typing methods correlate with virulence differences, with A1b strains more virulent than A1a, A2 and type B strains. These findings indicate type A strains are not equivalent with respect to virulence and have important implications for public health as well as basic research programs.     

Mouse Models of Aerosol-Acquired Tularemia Caused by Francisella tularensis Types A and B

After preliminary assessment of virulence in AKR/J, DBA/1, BALB/c, and C57BL/6 mice, we investigated histopathologic changes in BALB/c and C57BL/6 mice infected with type A (strain SCHU S4) or type B (strain 425) Francisella tularensis by aerosol exposure. In mice exposed to type A infection, changes in histologic presentation were not apparent until day 3 after infection, when pyogranulomatous inflammation was detected in spleens and livers of BALB/c mice, and in lungs and spleens of C57BL/6 mice. Histopathologic changes were most severe and widespread in both mouse strains on day 5 after infection and seemed to completely resolve within 22 d of challenge. BALB/c mice were more resistant than C57BL/6 mice in lethal-dose calculations, but C57BL/6 mice cleared the infection more rapidly. Mice similarly challenged with type B F. tularensis also developed histopathologic signs of infection beginning on day 3. The most severe changes were noted on day 8 and were characterized by granulomatous or pyogranulomatous infiltrations of the lungs. Unlike type A infection, lesions due to type B did not resolve over time and remained 3 wk after infection. In type B, but not type A, infection we noted extensive inflammation of the heart muscle. Although no microorganisms were found in tissues of type A survivors beyond 9 d after infection, mice surviving strain 425 infection had a low level of residual infection at 3 wk after challenge. The histopathologic presentation of tularemia caused by F. tularensis types A and B in BALB/c and C57BL/6 mice bears distinct similarities to tularemia in humans.Abbreviations: LVS, live vaccine strain; SUBLN, submandibular lymph node; MESLN, mesenteric lymph nodeTularemia is an infectious disease caused by the gram-negative bacterium Francisella tularensis. Clinical disease was first seen in Japan in 1837, when people who ate meat from rabbits developed fever, chills, and glandular tumors.²⁷ In the United States, the disease was first described in 1911 by Dr Edward Francis, who observed a plague-like illness in ground squirrels. In 1914, the first human case in the United States was described in a butcher.⁴⁸ Dr Francis, for whom the microorganism was named, characterized the clinical signs, symptoms, and the mode of transmission after infection.Tularemia is one of the most pathogenic diseases known, causing disease after inoculation or inhalation of as few as 10 microorganisms.²⁷ The microorganism is infectious by many routes, including ingestion of contaminated food or water, through microabrasions or bites from various arthropods, or inhalation of the infectious agent. The species F. tularensis includes several subspecies including tularensis, holarctica, novidia, and mediaasiatica. The type A F. tularensis subspecies tularensis is highly virulent in humans and is the dominant species in North America. Type B F. tularensis subspecies holarctica (also called palearctica) is usually found in European countries and is less virulent.⁴⁵ Humans infected with either subspecies can develop an acute febrile illness.F. tularensis is a facultative intracellular pathogen and multiplies in macrophages. Target organs are associated with the reticuloendothelial system and include lymph nodes, lungs, spleen, liver, and kidneys. In human pneumonic tularemia, infection progresses systemically from the lungs to other organs and causes pathologic changes, primarily to the liver and spleen.^9,11,19,38 Humans infected by aerosol exposure also show hemorrhagic inflammation of the airways, which can progress to bronchopneumonia.⁴³ Alveolar spaces become filled with a mononuclear cell infiltrate. Pleuritis with adhesions and effusion and hilar lymphadenopathy are commonly found.³³ The fatality rate for untreated pneumonic infections is approximately 30% to 60% for type A F. tularensis and approximately 10% for infections with type B F. tularensis.⁴² Because of its high infectivity, morbidity, and mortality, this microorganism is classified as a Category A Select Agent. F. tularensis can cause natural infections in a variety of animals, including rabbits, cats, prairie dogs, voles, raccoons, squirrels, rats, lemmings, wild mice, and other species.^{1,3,4,12,30,35,40,49} Although the source of infection can be inapparent, outdoor-housed rhesus macaques can acquire tularemia.^15,37Animal models using virulent (for humans) strains are essential for the development of efficacious vaccines and evaluation of antimicrobials against pathogenic diseases affecting humans. Due to the virulence of this microorganism, most experimental infections in rodent model systems use the Live Vaccine Strain (LVS) to minimize the risk to laboratory workers,¹⁷ although many recent studies use the more virulent type B and type A strains. LVS can be lethal to mice but is attenuated in humans. LVS is lethal in naïve mice and causes pathologic reactions similar to those seen with virulent strains in humans.⁸ However, using an LVS model of infection is not without potential problems. First, dose lethality varies widely, depending on the route of LVS infection. Naïve mice are most sensitive to intraperitoneal challenge, followed by intravenous, intranasal, aerosol, and intradermal challenge. Furthermore, the LD₅₀ for LVS administered by aerosol is greater than 10³ microorganisms, whereas the LD₅₀ for virulent F. tularensis strains is less than 10 microorganisms.⁸To better understand the pathogenesis of this important Category A pathogen, we determined the virulence and investigated the histopathogenesis of type A and type B F. tularensis in mice challenged by small-particle aerosol. We compared the histopathogenesis of infection over a period of 3 wk in BALB/c and C57BL/6 mice challenged with 1468 microorganisms or 100 microorganisms of F. tularensis SCHU S4 (type A). Type B (strain 425) infection was similarly investigated in BALB/c mice challenged with 102 microorganisms. Bacterial burdens in spleens, livers, lungs, and blood were determined.     

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.     

A ΔclpB Mutant of Francisella tularensis Subspecies holarctica Strain,FSC200, Is a More Effective Live Vaccine than F. tularensis LVS in a Mouse Respiratory Challenge Model of Tularemia

Francisella tularensis subsp. tularensis is a highly virulent pathogen for humans especially if inhaled. Consequently, it is considered to be a potential biothreat agent. An experimental vaccine, F. tularensis live vaccine strain, derived from the less virulent subsp. holarctica, was developed more than 50 years ago, but remains unlicensed. Previously, we developed a novel live vaccine strain, by deleting the chaperonin clpB gene from F. tularensis subsp. tularensis strain, SCHU S4. SCHU S4ΔclpB was less virulent for mice than LVS and a more effective vaccine against respiratory challenge with wild type SCHU S4. In the current study, we were interested to determine whether a similar mutant on the less virulent subsp. holarctica background would also outperform LVS in terms of safety and efficacy. To this end, clpB was deleted from clinical holarctica strain, FSC200. FSC200ΔclpB had a significantly higher intranasal LD₅₀ than LVS for BALB/c mice, but replicated to higher numbers at foci of infection after dermal inoculation. Moreover, FSC200ΔclpB killed SCID mice more rapidly than LVS. However, dermal vaccination of BALB/c mice with the former versus the latter induced greater protection against respiratory challenge with SCHU S4. This increased efficacy was associated with enhanced production of pulmonary IL-17 after SCHU S4 challenge.     

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.     

Use of Transposon-Transposase Complexes To Create Stable Insertion Mutant Strains of Francisella tularensis LVS

Francisella tularensis is a highly virulent zoonotic bacterial pathogen capable of infecting numerous different mammalian species, including humans. Elucidation of the pathogenic mechanisms of F. tularensis has been hampered by a lack of tools to genetically manipulate this organism. Herein we describe the use of transposome complexes to create insertion mutations in the chromosome of the F. tularensis live vaccine strain (LVS). A Tn5-derived transposon encoding kanamycin resistance and lacking a transposase gene was complexed with transposase enzyme and transformed directly into F. tularensis LVS by electroporation. An insertion frequency of 2.6 × 10⁻⁸ ± 0.87 × 10⁻⁸ per cell was consistently achieved using this method. There are 178 described Tn5 consensus target sites distributed throughout the F. tularensis genome. Twenty-two of 26 transposon insertions analyzed were within known or predicted open reading frames, but none of these insertions was associated with the Tn5 target site. Analysis of the insertions of sequentially passed strains indicated that the transposons were maintained stably at the initial insertion site after more than 270 generations. Therefore, transformation by electroporation of Tn5-based transposon-transposase complexes provided an efficient mechanism for generating random, stable chromosomal insertion mutations in F. tularensis.     

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.     

Inhibition of Clostridium botulinum by Strains of Clostridium perfringens Isolated from Soil

Thirty-one soil samples were examined for the presence of organisms capable of inhibiting growth and toxin production of strains of Clostridium botulinum type A. Such organisms were found in eight samples of soil. Inhibiting strains of C. perfringens were found in five samples, of C. sporogenes in three and of Bacillus cereus in three. Three of the C. perfringens strains produced an inhibitor effective on all 11 strains of C. botulinum type A against which they were tested, seven of eight proteolytic type B strains, one nonproteolytic type B strain, five of nine type E strains and all seven type F strains, whether proteolytic or nonproteolytic. They did not inhibit any of 26 type C strains, 6 type D strains, 4 type E strains, or 24 C. sporogenes strains. In mixed culture, an inhibitor strain of C. perfringens repressed growth and toxin production by a C. botulinum type A strain even though it was outnumbered by the latter about 40 times. It also repressed growth and toxin production of C. botulinum in mixed culture of soils in which this latter organism naturally occurred when cooked meat medium but not when trypticase medium was used.     

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.     

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.     

The type IV pilin,PilA, is required for full virulence of <Emphasis Type="Italic">Francisella tularensis</Emphasis> subspecies <Emphasis Type="Italic">tularensis</Emphasis>

Background  

All four Francisella tularensis subspecies possess gene clusters with potential to express type IV pili (Tfp). These clusters include putative pilin genes, as well as pilB, pilC and pilQ, required for secretion and assembly of Tfp. A hallmark of Tfp is the ability to retract the pilus upon surface contact, a property mediated by the ATPase PilT. Interestingly, out of the two major human pathogenic subspecies only the highly virulent type A strains have a functional pilT gene.     

Alternative Activation of Macrophages and Induction of Arginase Are Not Components of Pathogenesis Mediated by Francisella Species

Virulent Francisella tularensis ssp tularensis is an intracellular, Gram negative bacterium that causes acute lethal disease following inhalation of fewer than 15 organisms. Pathogenicity of Francisella infections is tied to its unique ability to evade and suppress inflammatory responses in host cells. It has been proposed that induction of alternative activation of infected macrophages is a mechanism by which attenuated Francisella species modulate host responses. In this report we reveal that neither attenuated F. tularensis Live Vaccine Strain (LVS) nor virulent F. tularensis strain SchuS4 induce alternative activation of macrophages in vitro or in vivo. LVS, but not SchuS4, provoked production of arginase1 independent of alternative activation in vitro and in vivo. However, absence of arginase1 did not significantly impact intracellular replication of LVS or SchuS4. Together our data establish that neither induction of alternative activation nor expression of arginase1 are critical features of disease mediated by attenuated or virulent Francisella species.     

Rapid High Resolution Genotyping of Francisella tularensis by Whole Genome Sequence Comparison of Annotated Genes (“MLST+”)

The zoonotic disease tularemia is caused by the bacterium Francisella tularensis. This pathogen is considered as a category A select agent with potential to be misused in bioterrorism. Molecular typing based on DNA-sequence like canSNP-typing or MLVA has become the accepted standard for this organism. Due to the organism’s highly clonal nature, the current typing methods have reached their limit of discrimination for classifying closely related subpopulations within the subspecies F. tularensis ssp. holarctica. We introduce a new gene-by-gene approach, MLST⁺, based on whole genome data of 15 sequenced F. tularensis ssp. holarctica strains and apply this approach to investigate an epidemic of lethal tularemia among non-human primates in two animal facilities in Germany. Due to the high resolution of MLST⁺ we are able to demonstrate that three independent clones of this highly infectious pathogen were responsible for these spatially and temporally restricted outbreaks.     

Francisella tularensis Type A Strains Cause the Rapid Encystment of Acanthamoeba castellanii and Survive in Amoebal Cysts for Three Weeks Postinfection

Francisella tularensis, the causative agent of the zoonotic disease tularemia, has recently gained increased attention due to the emergence of tularemia in geographical areas where the disease has been previously unknown and to the organism''s potential as a bioterrorism agent. Although F. tularensis has an extremely broad host range, the bacterial reservoir in nature has not been conclusively identified. In this study, the ability of virulent F. tularensis strains to survive and replicate in the amoeba Acanthamoeba castellanii was explored. We observe that A. castellanii trophozoites rapidly encyst in response to F. tularensis infection and that this rapid encystment phenotype is caused by factor(s) secreted by amoebae and/or F. tularensis into the coculture medium. Further, our results indicate that in contrast to the live vaccine strain LVS, virulent strains of F. tularensis can survive in A. castellanii cysts for at least 3 weeks postinfection and that the induction of rapid amoeba encystment is essential for survival. In addition, our data indicate that pathogenic F. tularensis strains block lysosomal fusion in A. castellanii. Taken together, these data suggest that interactions between F. tularensis strains and amoebae may play a role in the environmental persistence of F. tularensis.Francisella tularensis is the etiological agent of the zoonotic disease tularemia, also known as rabbit fever (35, 53). Strains belonging to F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, which are both prevalent in the Northern Hemisphere, cause the majority of reported cases of tularemia (36). Subspecies tularensis is highly contagious, with an infectious dose of 1 to 10 bacteria, and is associated with more severe disease (21). Though described more than a century ago as a disease common among hunters and trappers, tularemia has recently been reported in areas with no previous known risk (20, 25, 31, 42). F. tularensis infects a broad range of wildlife species (36), and a number of arthropods, such as ticks and flies, are known to be vectors (36, 49). Humans are usually infected either through an insect bite or by inhalation of aerosolized bacteria (49). Tularemia can be fatal in up to 30% of untreated cases (36, 49), with the mortality rate reaching 90% in pneumonic infections, as described in early studies conducted with vaccinated human volunteers (44-46, 49). Due to its highly infectious nature and its potential for use as a bioterrorism agent, F. tularensis has been classified as a class A biothreat pathogen by the Centers for Disease Control and Prevention (CDC), which has mandated that human tularemia be a reportable disease since 2000 (15, 37). In addition, the absence of a licensed vaccine for prophylaxis (36) makes understanding the virulence mechanisms used by this pathogen imperative for the development of efficacious measures to prevent or treat human disease.Though F. tularensis has been isolated from more than 250 wildlife species (21), the acute nature of the infections and the resultant high mortality rates in these hosts indicate that the bacterial reservoir(s) in nature have yet to be identified. Tularemia outbreaks involving F. tularensis subsp. holarctica have often been linked to water sources (6, 40), and a positive PCR field test was reported for Francisella during such an outbreak in Norway (5). Abd et al. reported that the F. tularensis live vaccine strain LVS is able to survive and replicate in the amoeba Acanthamoeba castellanii (1), suggesting a potential link between amoeba-Francisella interactions and environmental persistence. A. castellanii, a free-living environmental amoeba, is known to serve as a reservoir for a number of pathogenic microorganisms (24). However, to date, interactions of virulent F. tularensis subspecies tularensis strains with amoebae have not been documented. The ability of several human intracellular pathogens, including Legionella pneumophila and Mycobacterium avium, to infect and survive within amoebae has been well characterized (10, 12). In addition to playing a role in environmental survival and dissemination, growth in A. castellanii has been shown to enhance the ability of L. pneumophila and M. avium to survive and replicate in host macrophages (10, 12) and to enhance the virulence of both species in mice (7, 12). Since F. tularensis species are facultative intracellular pathogens that primarily survive in macrophages, probing the Francisella-amoeba interaction may provide insights into Francisella pathogenesis, as well as environmental survival. In this study, we investigated the ability of virulent type A strains of F. tularensis to survive in A. castellanii with a focus on understanding the role of Francisella-amoeba interactions in environmental persistence.     

Construction of a bioluminescence reporter plasmid for Francisella tularensis

A Francisella tularensis shuttle vector that constitutively expresses the Photorhabdus luminescens lux operon in type A and type B strains of F. tularensis was constructed. The bioluminescence reporter plasmid was introduced into the live vaccine strain of F. tularensis and used to follow F. tularensis growth in a murine intranasal challenge model in real-time by bioluminescence imaging. The results show that the new bioluminescence reporter plasmid represents a useful tool for tularemia research that is suitable for following F. tularensis growth in both in vitro and in vivo model systems.     

Description of two new plasmids isolated from Francisella philomiragia strains and construction of shuttle vectors for the study of Francisella tularensis

Francisella tularensis is the causative agent of tularemia, a zoonotic disease often transmitted to humans by infected animals. The lack of useful specific genetic tools has long hampered the study of F. tularensis subspecies. We identified and characterized two new plasmids, pF242 and pF243, isolated from Francisella philomiragia strains ATCC 25016 and ATCC 25017, respectively. Sequence analysis revealed that pF242 and pF243 are closely related to pC194 and pFNL10 plasmids, respectively. Two generations of pF242- and pF243-based shuttle vectors, harboring several antibiotic resistance markers, were developed. We used the first generation to compare transformation efficiencies in two virulent F. tularensis subspecies. We found that electroporation was more efficient than cryotransformation: almost all vectors tested were successfully introduced by electroporation into Francisella strains with a high level of efficiency. The second generation of shuttle vectors, containing a multiple cloning site and/or gfp gene downstream of Francisella groES promotor, was used for GFP production in F. tularensis. The development of new shuttle vectors offers new perspectives in the genetic manipulation of F. tularensis, helping to elucidate the mechanisms underlying its virulence.     

Comparative Resistance of Strains of Clostridium botulinum to Gamma Rays

A total of 102 strains of Clostridium botulinum (56 strains of type A, 43 type B, and 3 nontoxigenic strains which could not be typed) was examined for resistance to gamma rays. When these organisms were suspended in neutral phosphate buffer in concentrations of 10⁴ spores per tube, the threshold sterilizing dose appeared to be 1.4 Mrad. Partial survival to 1.4 Mrad was shown by 10.7% of the type A strains, 18.6% of the type B strains, and one of three nontoxigenic strains. Over-all, type A strains indicated higher radioresistance than type B strains, although there was overlapping. Representatives of the most resistant strains had D values of 0.317 to 0.336 Mrad; the D values of an intermediate group were 0.224 to 0.253 Mrad, and the most sensitive strain studied, 51B, had a D value of 0.129 Mrad. The radioresistance of Putrefactive Anaerobe 3679, strain S-2, was comparable to the intermediate C. botulinum group (D = 0.209).     

Nasal Acai Polysaccharides Potentiate Innate Immunity to Protect against Pulmonary Francisella tularensis and Burkholderia pseudomallei Infections

Pulmonary Francisella tularensis and Burkholderia pseudomallei infections are highly lethal in untreated patients, and current antibiotic regimens are not always effective. Activating the innate immune system provides an alternative means of treating infection and can also complement antibiotic therapies. Several natural agonists were screened for their ability to enhance host resistance to infection, and polysaccharides derived from the Acai berry (Acai PS) were found to have potent abilities as an immunotherapeutic to treat F. tularensis and B. pseudomallei infections. In vitro, Acai PS impaired replication of Francisella in primary human macrophages co-cultured with autologous NK cells via augmentation of NK cell IFN-γ. Furthermore, Acai PS administered nasally before or after infection protected mice against type A F. tularensis aerosol challenge with survival rates up to 80%, and protection was still observed, albeit reduced, when mice were treated two days post-infection. Nasal Acai PS administration augmented intracellular expression of IFN-γ by NK cells in the lungs of F. tularensis-infected mice, and neutralization of IFN-γ ablated the protective effect of Acai PS. Likewise, nasal Acai PS treatment conferred protection against pulmonary infection with B. pseudomallei strain 1026b. Acai PS dramatically reduced the replication of B. pseudomallei in the lung and blocked bacterial dissemination to the spleen and liver. Nasal administration of Acai PS enhanced IFN-γ responses by NK and γδ T cells in the lungs, while neutralization of IFN-γ totally abrogated the protective effect of Acai PS against pulmonary B. pseudomallei infection. Collectively, these results demonstrate Acai PS is a potent innate immune agonist that can resolve F. tularensis and B. pseudomallei infections, suggesting this innate immune agonist has broad-spectrum activity against virulent intracellular pathogens.