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

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

Common ancestry and novel genetic traits of Francisella novicida-like isolates from North America and Australia as revealed by comparative genomic analyses

Francisella novicida is a close relative of Francisella tularensis, the causative agent of tularemia. The genomes of F. novicida-like clinical isolates 3523 (Australian strain) and Fx1 (Texas strain) were sequenced and compared to F. novicida strain U112 and F. tularensis strain Schu S4. The strain 3523 chromosome is 1,945,310 bp and contains 1,854 protein-coding genes. The strain Fx1 chromosome is 1,913,619 bp and contains 1,819 protein-coding genes. NUCmer analyses revealed that the genomes of strains Fx1 and U112 are mostly colinear, whereas the genome of strain 3523 has gaps, translocations, and/or inversions compared to genomes of strains Fx1 and U112. Using the genome sequence data and comparative analyses with other members of the genus Francisella, several strain-specific genes that encode putative proteins involved in RTX toxin production, polysaccharide biosynthesis/modification, thiamine biosynthesis, glucuronate utilization, and polyamine biosynthesis were identified. The RTX toxin synthesis and secretion operon of strain 3523 contains four open reading frames (ORFs) and was named rtxCABD. Based on the alignment of conserved sequences upstream of operons involved in thiamine biosynthesis from various bacteria, a putative THI box was identified in strain 3523. The glucuronate catabolism loci of strains 3523 and Fx1 contain a cluster of nine ORFs oriented in the same direction that appear to constitute an operon. Strains U112 and Schu S4 appeared to have lost the loci for RTX toxin production, thiamine biosynthesis, and glucuronate utilization as a consequence of host adaptation and reductive evolution. In conclusion, comparative analyses provided insights into the common ancestry and novel genetic traits of these strains.     

Active suppression of the pulmonary immune response by Francisella tularensis Schu4

Francisella tularensis is an obligate, intracellular bacterium that causes acute, lethal disease following inhalation. As an intracellular pathogen F. tularensis must invade cells, replicate, and disseminate while evading host immune responses. The mechanisms by which virulent type A strains of Francisella tularensis accomplish this evasion are not understood. Francisella tularensis has been shown to target multiple cell types in the lung following aerosol infection, including dendritic cells (DC) and macrophages. We demonstrate here that one mechanism used by a virulent type A strain of F. tularensis (Schu4) to evade early detection is by the induction of overwhelming immunosuppression at the site of infection, the lung. Following infection and replication in multiple pulmonary cell types, Schu4 failed to induce the production of proinflammatory cytokines or increase the expression of MHCII or CD86 on the surface of resident DC within the first few days of disease. However, Schu4 did induce early and transient production of TGF-beta, a potent immunosuppressive cytokine. The absence of DC activation following infection could not be attributed to the apoptosis of pulmonary cells, because there were minimal differences in either annexin or cleaved caspase-3 staining in infected mice compared with that in uninfected controls. Rather, we demonstrate that Schu4 actively suppressed in vivo responses to secondary stimuli (LPS), e.g., failure to recruit granulocytes/monocytes and stimulate resident DC. Thus, unlike attenuated strains of F. tularensis, Schu4 induced broad immunosuppression within the first few days after aerosol infection. This difference may explain the increased virulence of type A strains compared with their more attenuated counterparts.     

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.     

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.     

Towards proteome database of Francisella tularensis

The accessibility of the partial genome sequence of Francisella tularensis strain Schu 4 was the starting point for a comprehensive proteome analysis of the intracellular pathogen F. tularensis. The main goal of this study is identification of protein candidates of value for the development of diagnostics, therapeutics and vaccines. In this review, the current status of 2-DE F. tularensis database building, approaches used for identification of biologically important subsets of F. tularensis proteins, and functional and topological assignments of identified proteins using various prediction programs and database homology searches are presented.     

A new cell assay to determine the virulence of Francisella tularensis

A cell culture assay to determine the virulence of Francisella tularensis was devised. Murine cell lines P388 and J774 were significantly more susceptible to F. tularensis Schu4 than the attenuated live vaccine strain. The ability of F. tularensis strains to cause cell death correlated with their virulence to mice. Use of this assay with infected cells separated from susceptible uninfected cells by a membrane with 0.1 μm pores, failed to demonstrate possible diffusible exotoxins produced by F. tularensis.     

Chromosome rearrangement and diversification of Francisella tularensis revealed by the type B (OSU18) genome sequence

The gamma-proteobacterium Francisella tularensis is one of the most infectious human pathogens, and the highly virulent organism F. tularensis subsp. tularensis (type A) and less virulent organism F. tularensis subsp. holarctica (type B) are most commonly associated with significant disease in humans and animals. Here we report the complete genome sequence and annotation for a low-passage type B strain (OSU18) isolated from a dead beaver found near Red Rock, Okla., in 1978. A comparison of the F. tularensis subsp. holarctica sequence with that of F. tularensis subsp. tularensis strain Schu4 (P. Larsson et al., Nat. Genet. 37:153-159, 2005) highlighted genetic differences that may underlie different pathogenicity phenotypes and the evolutionary relationship between type A and type B strains. Despite extensive DNA sequence identity, the most significant difference between type A and type B isolates is the striking amount of genomic rearrangement that exists between the strains. All but two rearrangements can be attributed to homologous recombination occurring between two prominent insertion elements, ISFtu1 and ISFtu2. Numerous pseudogenes have been found in the genomes and are likely contributors to the difference in virulence between the strains. In contrast, no rearrangements have been observed between the OSU18 genome and the genome of the type B live vaccine strain (LVS), and only 448 polymorphisms have been found within non-transposase-coding sequences whose homologs are intact in OSU18. Nonconservative differences between the two strains likely include the LVS attenuating mutation(s).     

Characterization of Francisella tularensis outer membrane proteins

Francisella tularensis is a gram-negative coccobacillus that is capable of causing severe, fatal disease in a number of mammalian species, including humans. Little is known about the proteins that are surface exposed on the outer membrane (OM) of F. tularensis, yet identification of such proteins is potentially fundamental to understanding the initial infection process, intracellular survival, virulence, immune evasion and, ultimately, vaccine development. To facilitate the identification of putative F. tularensis outer membrane proteins (OMPs), the genomes of both the type A strain (Schu S4) and type B strain (LVS) were subjected to six bioinformatic analyses for OMP signatures. Compilation of the bioinformatic predictions highlighted 16 putative OMPs, which were cloned and expressed for the generation of polyclonal antisera. Total membranes were extracted from both Schu S4 and LVS by spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation, which separated OMs from cytoplasmic (inner) membrane and other cellular compartments. Validation of OM separation and enrichment was confirmed by probing sucrose gradient fractions with antibodies to putative OMPs and inner membrane proteins. F. tularensis OMs typically migrated in sucrose gradients between densities of 1.17 and 1.20 g/ml, which differed from densities typically observed for other gram-negative bacteria (1.21 to 1.24 g/ml). Finally, the identities of immunogenic proteins were determined by separation on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This is the first report of a direct method for F. tularensis OM isolation that, in combination with computational predictions, offers a more comprehensive approach for the characterization of F. tularensis OMPs.     

Characterization of the siderophore of Francisella tularensis and role of fslA in siderophore production

We determined that LVS and Schu S4 strains of the human pathogen Francisella tularensis express a siderophore when grown under iron-limiting conditions. We purified this siderophore by conventional column chromatography and high-pressure liquid chromatography and used mass spectrometric analysis to demonstrate that it is structurally similar to the polycarboxylate siderophore rhizoferrin. The siderophore promoted the growth of LVS and Schu S4 strains in iron-limiting media. We identified a potential siderophore biosynthetic gene cluster encoded by fslABCD in the F. tularensis genome. The first gene in the cluster, fslA, encodes a member of the superfamily of nonribosomal peptide synthetase-independent siderophore synthetases (NIS synthetases) characterized by the aerobactin synthetases IucA and IucC. We determined that fslA is transcribed as part of an operon with downstream gene fslB and that the expression of the locus is induced by iron starvation. A targeted in-frame nonpolar deletion of fslA in LVS resulted in the loss of siderophore expression and in a reduced ability of F. tularensis to grow under conditions of iron limitation. Siderophore activity and the ability to grow under iron limitation could be regained by introducing the fslA(+) gene on a complementing plasmid. Our results suggest that the fslA-dependent siderophore is important for survival of F. tularensis in an iron-deficient environment.     

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.     

Identification of differentially regulated francisella tularensis genes by use of a newly developed Tn5-based transposon delivery system

Francisella tularensis is the etiologic agent of an intracellular systemic infection of the lymphatic system in humans called tularemia. The organism has become the subject of considerable research interest due to its classification as a category A select agent by the CDC. To aid genetic analysis of this pathogen, we have constructed a temperature-sensitive Tn5-based transposon delivery system that is capable of generating chromosomal reporter fusions with lacZ or luxCDABE, enabling us to monitor gene expression. Transposition is catalyzed by the hyperactive Tn5 transposase, whose expression is driven by the Francisella groES promoter. When high-temperature selection (42 degrees C) is applied to a bacterial culture carrying the transposon delivery plasmid, approximately 0.1% of the population is recovered with Tn5 insertions in the chromosome. Nucleotide sequence analysis of a sample of mutants revealed that the insertions occur randomly throughout the chromosome. The kanamycin-selectable marker of the transposon is also flanked by FLP recombination target sequences that allow deletion of the antibiotic resistance gene when desired. This system has been used to generate transposon mutant libraries for the F. tularensis live vaccine strain as well as two different virulent F. tularensis strains. Chromosomal reporters delivered with the transposon were used to identify genes upregulated by growth in Chamberlain's defined medium. Genes in the fsl operon, reported to be involved in iron acquisition, as well as genes in the igl gene cluster were among those identified by the screen. Further experiments implicate the ferric uptake regulator (Fur) protein in the negative regulation of fsl but not igl reporters, which occurs in an iron-dependent manner. Our results indicate that we have created a valuable new transposon that can be used to identify and characterize virulence genes in F. tularensis strains.     

Targeted inactivation of francisella tularensis genes by group II introns

Studies of the molecular mechanisms of pathogenesis of Francisella tularensis, the causative agent of tularemia, have been hampered by a lack of genetic techniques for rapid targeted gene disruption in the most virulent subspecies. Here we describe efficient targeted gene disruption in F. tularensis utilizing mobile group II introns (targetrons) specifically optimized for F. tularensis. Utilizing a targetron targeted to blaB, which encodes ampicillin resistance, we showed that the system works at high efficiency in three different subspecies: F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, and "F. tularensis subsp. novicida." A targetron was also utilized to inactivate F. tularensis subsp. holarctica iglC, a gene required for virulence. The iglC gene is located within the Francisella pathogenicity island (FPI), which has been duplicated in the most virulent subspecies. Importantly, the iglC targetron targeted both copies simultaneously, resulting in a strain mutated in both iglC genes in a single step. This system will help illuminate the contributions of specific genes, and especially those within the FPI, to the pathogenesis of this poorly studied organism.     

Outer membranes of a lipopolysaccharide-protein complex (LPS-17 kDa protein) as chemical tularemia vaccines

Abstract Immunisation with outer membranes of Francisella tularensis induced an efficient protection in guinea pigs against challenge with the virulent strains 503 or 144/713 (type B biovar holarctica ), both clinical isolates, and prevented the development of typical signs of infection in hamadryads (baboons), challenged with the virulent strain Schu (type A, biovar tularensis ) of F. tularensis . Immunisation with a lipopolysaccharide protein complex isolated from the outer membranes afforded protection in CBA mice against challenge with strain 503. Another LPS-protein complex obtained by the simple mixture of LPS preparations from strain 503 and a 17-kDa membrane protein from the avirulent R-variant of the vaccine strain 15 also demonstrated protective properties against experimental tularemia in mice.     

Proteome analysis of the purine stimulon from Lactococcus lactis

A comparative expression proteome analysis was carried out by analyzing differential expression patterns of pulse-labelled proteins on two-dimensional gels under standard conditions and during purine nucleotide starvation, followed by mass spectrometric identification of regulated proteins. Based upon the expression patterns, three stimulons could be identified in Lactococcus lactis subsp. cremoris. The Psu proteins (purine starvation up-regulated) had increased synthesis during purine depletion in a purine auxotroph. Among these proteins were enzymes of the purine biosynthesis pathways (PurE, PurS, PurM, PurL), and enzymes involved in the generation of C1 units (GlyA, Fhs). C1 units are primarily required for purine biosynthesis. Upon analysis of the nucleotide sequence preceding the structural genes for these proteins in the L. lactis IL1403 genome sequence showed that all contained PurBox-Pribnov box structures resembling the PurR activated promoters for the purDEK and purCSQLF operons. Most, and possibly all members of the Psu stimulon are thus members of the PurR regulon. Five Psu proteins could not be identified. The second stimulon, the Psd stimulon (purine starvation decreased), whose members are down-regulated during purine depletion, contained proteins related to protein synthesis (PpsB, EF-TS, trigger factor), or to GTPases (FtsZ, EF-TS); or are involved in energy metabolism (GapB, CcpA). No common regulatory elements could be found for members of this stimulon. Two Psd proteins escaped identification. The last, Dcu (decoynine up-regulated), stimulon contained proteins whose synthesis escaped the severe general depression during inhibition of the GMP synthetase by decoynine. This regulon was comprised of mostly glycolytic enzymes (fructose bisphosphate aldolase, enolase, pyruvate kinase) and translation elongation factors (GTPases: EF-TU, EF-G). Two Dcu proteins could not be identified. Out of 28 proteins subjected to mass spectrometry, 19 could be readily identified despite the fact that only the genome sequence of a strain of L. lactis subsp. lactis was available. The two subspecies share about 85% sequence identity, comparable to the genetic distance between Escherichia coli and Salmonella typhimurium. A success rate of 68% indicates that it may be feasible to perform proteomics based upon genomic sequences of relatives outside the genus.