J Med Microbiol 57 (2008), 1183-1192; DOI: 10.1099/jmm.0.2008/001826-0
© 2008 Society for General Microbiology
ISSN 1473-5644
An intracellularly inducible gene involved in virulence and polyphosphate production in Francisella
Mark I. Richards1,
Stephen L. Michell2 and
Petra C. F. Oyston1
1 Microbiology, Dstl Porton Down, Salisbury, Wiltshire SP4 0JQ, UK
2 School of Biosciences, University of Exeter, Exeter EX4 4QJ, UK
Correspondence
Petra C. F. Oyston
pcoyston{at}dstl.gov.uk
Received 10 March 2008
Accepted 11 June 2008
Francisella tularensis is an intracellular pathogen capable of multiplying to high levels in macrophages. By protein analysis, only a few proteins have been shown previously to be expressed at high levels in macrophages relative to bacteria grown in culture media. To identify additional genes that show increased expression during intracellular growth, we developed a plasmid for use in Francisella based on the induction of expression of green fluorescent protein. Clones of F. tularensis subsp. novicida were identified that were fluorescent only intracellularly and not when grown in vitro. Sequencing identified a range of genes comprising some such as dnaK that are already known to be expressed intracellularly and some novel targets. One of these newly identified regulated genes, FTN1472/FTT1564, was selected for further study. Isogenic mutants were generated in F. tularensis subsp. novicida and subsp. tularensis by allelic replacement. Inactivation of the gene resulted in abolition of polyphosphate production by F. novicida, strongly supporting the bioinformatic analysis, which had suggested that the gene may encode a polyphosphate kinase. The mutants exhibited defects for intracellular growth in macrophages and were attenuated in mice, indicating a key role for the putative polyphosphate kinase in the virulence of Francisella.
Abbreviations: GFP, green fluorescent protein; i.p., intraperitoneal; LD50, median lethal dose to induce morbidity or death; s.c., subcutaneous.
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INTRODUCTION
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Francisella tularensis is the causative agent of the zoonotic disease tularaemia. There are four subspecies of F. tularensis, differing in their virulence for man: F. tularensis subsp. tularensis is the most virulent for man, subsp. holarctica and mediasiatica are moderately virulent, and subsp. novicida is relatively avirulent (Oyston et al., 2004). The presentation of disease depends on the route of infection (Ellis et al., 2002). Typhoidal and respiratory tularaemia are the most serious forms of disease and have high associated mortality rates. Although infection can be treated with appropriate antibiotics, there is no vaccine available for prophylaxis. An undefined attenuated Live Vaccine Strain (LVS) was identified during the last century, but as yet this strain has failed to achieve regulatory approval.
F. tularensis is fastidious, requiring a rich medium supplemented with cysteine for growth in the laboratory, but it is primarily an intracellular pathogen, able to grow to high numbers within a wide range of cell types. However, invasion and proliferation in most cell types does not occur to the same level as is observed in macrophages and amoebae. Although intracellular multiplication is essential for pathogenicity of the bacterium, very little is known about the intracellular lifestyle of this organism, hindered in part by the lack of genetic tools available for working with Francisella.
Proteomic studies have revealed upregulation of very few proteins during intracellular growth (Golovliov et al., 1997). Although the molecular mechanisms of pathogenesis are not well understood, replication inside macrophages is essential for the organism to cause disease (Fortier et al., 1994). Until recently, only a limited number of genes had been shown to be required for intracellular growth, most notably the Francisella pathogenicity island carrying the igl genes (Golovliov et al., 2003; Lauriano et al., 2004; Lindgren et al., 2004; Twine et al., 2005). More recently, as genetic tools have been developed for use with F. tularensis (LoVullo et al., 2006), and aided by the publication of the genome sequence (Larsson et al., 2005), other genes involved in intracellular growth have been identified (Maier et al., 2007; Pechous et al., 2006; Tempel et al., 2006). In this study, we undertook promoter trapping to identify Francisella genes induced intracellularly. This allowed the identification of a gene encoding polyphosphate kinase, which we showed to be required for intracellular growth and virulence.
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METHODS
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Bacterial strains and culture conditions.
The bacterial strains used in this study are listed in Table 1
. F. tularensis strains were grown routinely on blood cysteine glucose agar (BCGA) containing 4 % cysteine, 4 % histidine, 5 % glucose and 10 % fresh filtered horse blood, solidified Chamberlain’s defined medium (CDM) (Chamberlain, 1965) or Thayer–Martin agar (Thomas et al., 2007). Where required, the medium was supplemented with 100 µg polymixin ml–1 and 10 µg chloramphenicol ml–1. Escherichia coli strains were grown in Luria–Bertani (LB) medium or on LB agar at 37 °C, supplemented as required with 30 µg chloramphenicol ml–1. All work undertaken with Francisella strains was performed under appropriate laboratory containment conditions in accordance with relevant legislative requirements.
Plasmids and manipulation of DNA.
Generally, the manipulation of DNA was carried out as outlined previously (Sambrook et al., 1989). Genomic DNA was isolated using a Puregene DNA Isolation kit (Gentra Systems). Plasmid DNA was isolated using Qiagen Mini and Maxi Plasmid kits. The plasmids used in this study are shown in Table 1
. Southern blotting was performed under high-stringency conditions as described by Sambrook et al. (1989). The oligonucleotide primers used for PCR in this study are given in Table 1. A standard PCR was performed with 25 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 30 s.
Construction of the promoter trap vector.
The plasmid pKK202 is stably maintained in F. novicida (Norqvist et al., 1996). To facilitate conjugation, the oriT locus was subcloned into pKK202 from pPV2 (Golovliov et al., 2003) to create pKK202oriT. To release the oriT locus, pPV2 was digested with BamHI and the 1.7 kb fragment was isolated following agarose gel electrophoresis. The fragment was ligated into pKK202 that had been linearized with BclI. The gene encoding green fluorescent protein (GFP) was amplified from pGFPuv (Clontech) by PCR using primers GFPFor/GFPRev (Table 1). The PCR product was ligated into pGEM-T Easy according to the manufacturer’s instructions. The oligonucleotide primers were designed to include ApaLI restriction sites, allowing the insert to be isolated from the pGEM-T Easy vector and cloned into pKK202oriT linearized with ApaLI. This plasmid was designated pKKoriTGFP. One of the oligonucleotide primers used to amplify the gene from pGFPuv was designed to incorporate an FbaI site directly upstream. Digestion with FbaI produces overhangs compatible with those produced by Sau3AI. As FbaI is methylation-sensitive, the plasmid was transformed into E. coli SCS110 to produce an unmethylated plasmid preparation.
Production of a genomic library.
Genomic DNA was isolated from F. novicida strain U112 and partially digested with Sau3AI (Roche). The DNA fragments were separated on a 0.7 % agarose gel and a region covering <2 kb was selected. The DNA was purified from the gel and ligated into pKKoriTGFP, which had been linearized with the restriction enzyme FbaI. This process was repeated several times to generate different sublibraries. The plasmids were introduced into E. coli S17
pir by electroporation for conjugation into F. novicida U112. Clones were selected on Thayer–Martin agar supplemented with chloramphenicol (to identify clones containing the plasmid) and polymixin (to inhibit growth of the E. coli donor). Clones were picked onto BCGA medium supplemented with chloramphenicol and in duplicate onto LB+chloramphenicol agar. Colonies growing on LB+chloramphenicol agar were examined under UV illumination and fluorescent colonies were removed from the library.
Macrophage assays.
The J774 mouse macrophage cell line was maintained in Dulbecco’s modified Eagle’s medium (Gibco) and incubated at 37 °C in 5 % CO2. The macrophages were passaged a maximum of 25 times. Bacterial cells were resuspended in 3 ml L-15 medium containing GlutaMAX and 10 % fetal calf serum (Life Technologies). The OD600 of this suspension was measured and the suspension was suitably diluted to give an approximate concentration of 1x109 c.f.u. ml–1. The L-15 medium was aspirated from each well and 100 µl bacterial suspension was overlaid onto the macrophages to give an m.o.i. of 10 bacteria per macrophage. Control wells were overlaid with L-15 medium alone. The cells were incubated for 30 min at 37 °C, after which time the medium was replaced with L-15 containing 10 µg gentamicin ml–1. The cells were incubated at 37 °C for 30 min. The medium was then removed and replaced with L-15 containing 2 µg gentamicin ml–1. To identify intracellularly expressed promoters, macrophages were examined at intervals up to 24 h for fluorescence using a confocal microscope (Olympus Instruments). Fluorescent pools were broken down into single clones to identify the fluorescent clones.
To follow bacterial survival, at selected intervals two wells of infected macrophages per strain were washed twice with 1 ml PBS, and 1 ml dH2O was added to each well. The macrophages were disrupted by aspiration 30 times. The cell lysate was serially diluted in PBS and dilutions were plated onto BCGA, which were incubated at 37 °C for 2 days. At the final sampling time, the uninfected controls were similarly processed and plated to detect possible contamination. A two-way analysis of variance was performed with Bonferroni’s post-test using GraphPad Prism v4.0.
Generation of 
1564 : : CAM mutants in subsp. novicida and tularensis.
Primers were designed from the genome sequence of F. tularensis subsp. tularensis strain SCHU S4 to generate an 831 bp deletion in the FTT1564 ORF as follows. Primers 1564LFF/1564LFR and 1564RFF/1564RFR were used to amplify DNA regions flanking the deletion. These were ligated into pGEM-T Easy according to the manufacturer’s protocol to generate p1564L and pGEM1564R containing the left and right flanks, respectively. The plasmids were digested with BglII and XhoI to linearize p1564L and to isolate the insert from p1564R. The p1564R insert was then ligated into p1564L to generate pGEM-T Easy containing both left and right flanks, designated p1564LR. The internal BglII restriction site between the two flanks allowed the insertion of a chloramphenicol resistance cassette (Quarry et al., 2007). The plasmid was digested with MluI and the insert was isolated and ligated into similarly digested pSMP22 to produce pMR1564 : : CAM. Plasmid pMR1564 : : CAM was introduced into F. tularensis subsp. novicida U112 or subsp. tularensis SCHU S4 by conjugation using the method of Golovliov et al. (2003). Briefly, E. coli S17pir pMR1564 : : CAM was grown overnight in LB+chloramphenicol; a 1 ml aliquot was removed and the bacteria were pelleted by centrifugation and resuspended in 50 µl fresh LB broth. Francisella was grown on BCGA overnight at 37 °C in a confluent lawn. Bacteria were removed using a sterile loop, resuspended in the E. coli suspension and the mixture was spotted onto BCGA. After incubation overnight at 25 °C, the bacteria were removed with a sterile loop and resuspended in 500 µl PBS before being inoculated onto Thayer–Martin agar supplemented with 100 µg polymixin ml–1 (to inhibit growth of the E. coli) and 10 µg chloramphenicol ml–1. Colonies were subcultured onto Thayer–Martin agar supplemented with 5 % sucrose and 10 µg chloramphenicol ml–1. Isolated colonies that grew in the presence of sucrose were analysed by PCR for the deletion of the FTN1472/FTT1564 ORF and loss of the vector (mutants U112FTN1472 : : CAM and SCHUS4FTT1564 : : CAM, respectively). This was subsequently confirmed by Southern blotting.
Animal studies.
Groups of six 6–8-week-old female BALB/c mice (Charles River Laboratories) were dosed by the intraperitoneal (i.p.) or subcutaneous (s.c.) route with serial dilutions of bacteria in PBS. The concentration of bacteria in the inoculum was determined by viable count. Mice surviving the initial challenge were subsequently challenged 56 days later with wild-type strains to determine whether a protective immune response had been induced. Humane end points were strictly observed and animals deemed incapable of survival were killed humanely by cervical dislocation. The median lethal dose to induce morbidity or death (LD50) has been shown to be approximately 1 c.f.u. for SCHU S4 given by the s.c. route and for U112 by the i.p. route (Quarry et al., 2007), and 3x102 c.f.u. for U112 by the s.c. route (P. Oyston, personal communication).
Polyphosphate analysis.
Wild-type and mutant strains of F. novicida were grown overnight in CDM. The inorganic phosphate concentration in CDM is 5 mM. Bacteria were then subcultured into 28 ml pre-warmed CDM to a concentration of approximately 1.6x109 c.f.u. ml–1 (Milne et al., 2007) and incubated for a further 24 h, resulting in cultures with a density of 2–2.57x1010 c.f.u. ml–1. Polyphosphate extraction was performed using a modification of the acidic extraction method (Ghorbel et al., 2006). One millilitre aliquots of each bacterial culture were removed and the bacterial cells were pelleted by centrifugation. The pellet was resuspended in 1 ml 0.5 M HClO4 and incubated on ice for 30 min with occasional inversion. The bacterial cells were pelleted by centrifugation and the supernatant was retained for analysis. Nucleoside phosphates were removed by adsorption on acid-washed activated charcoal (Sigma). The free phosphates in the acid-soluble extract were determined before and after hydrolysis at 100 °C for 10 min. An increase in detectable free phosphate following hydrolysis is a measure of the amount of polyphosphate present in the bacteria. The concentration of free phosphate was determined using a spectrophotometric phosphorus assay kit based on phosphate complex formation with malachite green and molybdate (BioAssay Systems). The mean results from eight replicates were analysed by a two-way analysis of variance with Bonferroni’s post-tests using GraphPad Prism v4.0.
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RESULTS AND DISCUSSION
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Identification of a putative polyphosphate kinase expressed by Francisella within macrophages
F. tularensis has been described as a ‘stealth pathogen’ (Sjostedt, 2006) in that very little is known about how the organism causes disease. However, as an intracellular pathogen it has long been assumed that an inability to multiply within cells would cause attenuation, and that genes key for intracellular survival would help elucidate the basis of pathogenicity. The ability to identify such genes has been hampered firstly by the lack of genetic tools for use with Francisella and secondly by the difficulties encountered in transformation. The development of vectors capable of mobilization from E. coli into Francisella allows the efficient transfer of these shuttle plasmids. Due the paucity of genetic tools for use with Francisella, it was necessary to construct a suitable vector to facilitate this study. To this end, the plasmid pKKoriTGFP was developed (Fig. 1). The backbone of the vector was derived from pKK202, which is stably maintained in both E. coli and Francisella. As electroporation is poorly efficient in Francisella compared with conjugation, the oriT locus of pPV2 was inserted into pKK202 to produce a mobilizable vector. A promoter trapping vector based on induction of chloramphenicol acetyltransferase has been shown to be able to identify intracellularly induced genes (Kuoppa et al., 2001). The drawback of the use of antibiotic resistance as an inducible marker is that it requires the introduction of multiple resistance genes into a human pathogen. To reduce the dependence on multiple antibiotic resistance markers, we developed a promoter trapping vector, pKKoitTGFP, carrying a promoterless gene encoding the cycle 3 variant of GFP (Crameri et al., 1996). GFP has been used to identify genes induced in Erwinia chrysanthemi during plant infection (Yang et al., 2004), and GFP expression was used to identify genes expressed by Salmonella enterica serovar Typhimurium, Mycobacterium tuberculosis and Listeria monocytogenes during growth inside macrophages (Barker et al., 1998; Valdivia & Falkow, 1997; Wilson et al., 2001). However, because the detection of induced fluorescence was by visual inspection of macrophages by confocal microscopy, the pools in this study could not be as complex as an antibiotic selection approach would allow.
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Fig. 1. Plasmid pKKoriTGFP: a mobilizable plasmid suitable for use in Francisella, carrying a chloramphenicol-resistance cassette for plasmid stabilization and a promoterless gene encoding GFP. An FbaI restriction site immediately preceding the gene encoding GFP allowed insertion of Sau3AI genomic digest fragments.
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F. novicida is frequently used as a model for the more pathogenic subspecies, as the subspecies are genetically closely related and the mechanisms of intracellular survival are likely to be conserved in general (Brotcke et al., 2006). Not only is F. novicida safer to work with than the highly virulent strains, but it grows more quickly and is more amenable to genetic manipulation. However, extrapolating from F. novicida to the highly virulent subspecies must be undertaken with caution. It is apparent that the immune response required for protection against challenge with subsp. tularensis strains is different to that required for protection against F. novicida (Fulop et al., 2001; Shen et al., 2004). Despite this, studies in F. novicida are useful in identifying attenuation targets for further study in subsp. tularensis. Genomic DNA was isolated from F. tularensis subsp. novicida U112 and partially digested with Sau3AI. Fragments of <2 kb in size were ligated into the FbaI site of pKKoriTGFP. The pool of plasmids was transferred to F. tularensis subsp. novicida U112 by conjugation from E. coli S17pir and chloramphenicol-resistant colonies were isolated. Over 550 colonies were picked into gridded libraries. Of these, approximately 1 % fluoresced on agar when examined under UV light and were removed from the library. This is similar to the proportion of in vitro-expressed promoters identified by Kuoppa et al. (2001) when screening for cat activity. The non-fluorescent clones were assembled into pools of eight for screening in macrophages. It was found that increasing the complexity of the pools reduced the ability to detect fluorescent bacteria (data not shown). The infected macrophages were examined at 24 h for bacteria expressing GFP (Fig. 2). In total, 77 pools of mutants were screened in macrophages. Eighteen positive pools were then examined in pure culture to identify the intracellularly fluorescent clones and 23 fluorescent clones were identified. Inserts were sequenced and the DNA sequence was compared with the genome sequences for strain SCHU S4, the only published Francisella genome at the time. Subsequently, the F. tularensis subsp. novicida genome became available and the insert sequences were then compared with this as confirmation. This allowed the identification of intergenic regions that may encode intracellularly induced promoters (Table 2).
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Fig. 2. Macrophages infected with an F. novicida pKKoriTGFP pool containing a clone (subsequently identified as dnaK) with an intracellularly inducible promoter, at 24 h post-infection.
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Acid phosphatase (AcpA) and DnaK were identified in this study, and are both known to be expressed intracellularly. AcpA is proposed to be involved in inhibition of the respiratory burst (Reilly et al., 1996), but recently phospholipase activity has also been proposed for AcpA, with a role in phagosomal trafficking of the bacterium (Felts et al., 2006; Mohapatra et al., 2007). However, there are conflicting reports on whether AcpA is essential for virulence and intracellular replication (Baron et al., 1999; Mohapatra et al., 2007), which are yet to be resolved. DnaK was first demonstrated to be upregulated in macrophages using a proteomic approach (Golovliov et al., 1997) and was identified as being essential for intracellular growth and virulence of subsp. novicida (Tempel et al., 2006). Identification of these promoters already known to be induced intracellularly provided confirmation that this GFP-based approach could identify intracellularly inducible promoters.
Of the remaining genes identified as being induced intracellularly, none had been identified previously as being involved in the intracellular growth of Francisella. Two-component regulatory systems have been extensively studied for their contribution to intracellular growth. One promoter identified in this study was upstream of the kdpD gene. KdpD is the sensor of a two-component regulatory system, which in E. coli monitors K+ concentration and turgor pressure (Sugiura et al., 1994). An associated response regulator has not been reported in Francisella, but the kdpE homologue, encoding the cognate response regulator in E. coli, is a pseudogene in strains SCHU S4, LVS and OSU18.
One of the genes identified in this study was yfdH, predicted by sequence homology to encode a glycosyltransferase group 2 family protein. The same gene was identified during a genome comparison study as a potential cause of attenuation of the LVS strain (Rohmer et al., 2006), as it is a pseudogene in LVS but was intact in 15 further strains examined. The locus appeared to be under strong purifying selection, indicating that its product plays an important role in bacterial survival or fitness (Rohmer et al., 2006). The putative glycosyltransferase is predicted to be involved in cell wall biogenesis or LPS biosynthesis. The LPS of strain LVS lacks galactosamine 1-phosphate compared with the LPS of another subsp. holarctica strain, and the inactivation of yfdH may be the cause of this difference.
Two genes identified as having intracellularly inducible promoters, FTN0994/FTT0689 and FTN1472/FTT1564, were annotated as hypothetical proteins (Table 2). At the time they were identified, the proteins encoded by these genes had not been identified previously as having a role that may be important in vivo, either by microarray analysis (Brotcke et al., 2006; Deng et al., 2006) or by proteomics-based approaches (Twine et al., 2006). However, recently the FTN1472/FTT1564 homologue in the attenuated subsp. holarctica strain LVS (FTL0544) was identified as playing a role in intracellular survival and virulence (Maier et al., 2007). However, the suggested identity of the genes as encoding a polyphosphate kinase was not confirmed. Therefore, we wished to confirm whether the gene we had identified did in fact encode a polyphosphate kinase and determine what role this played in the virulence of F. tularensis subsp. tularensis and subsp. novicida.
FTN1472/FTT1564 is required for intracellular growth and virulence of F. tularensis
Polyphosphate has been shown to be key in determining how bacteria respond to stress, possibly by contributing to the production of ppGpp (Manganelli, 2007), and has been shown to be required for virulence for a range of pathogens including Pseudomonas aeruginosa (Rashid et al., 2000), Campylobacter jejuni (Candon et al., 2007), Salmonella enterica and Shigella flexneri (Kim et al., 2002). We therefore wished to determine what effect inactivation of this putative polyphosphate kinase had on the virulence of Francisella.
Mutants of F. tularensis subsp. tularensis and subsp. novicida were generated containing a deletion encompassing the majority of the FTN1472/FTT1564 ORF, replacing it with a chloramphenicol-resistance cassette. Examination of the genome sequence data for strains SCHU S4 (Larsson et al., 2005) and U112 (Rohmer et al., 2007) indicated that the FTT1564 gene was not part of an operon (Fig. 3) and that the observed phenotypes would therefore be unlikely to be due to polar effects on downstream genes. The plasmid was introduced into Francisella by conjugation, and mutants were selected by chloramphenicol resistance and sucrose tolerance. Replacement of the gene with the antibiotic-resistance cassette was confirmed by PCR and Southern blotting. The colony morphology of the mutants was identical to that of the wild-type strains and they grew to a similar density in liquid culture (data not shown). Bioinformatic analysis has suggested FTN1472/FTT1564 to be a polyphosphate kinase (Maier et al., 2007), but this function has not been proven. Therefore, we quantified the polyphosphate content of F. novicida U112 and the U112FTN1472 : : CAM mutant (Fig. 4). Polyphosphate is a linear polymer of hundreds of phosphate residues, found in bacterial, fungal, plant and animal cells (Kulaev & Kulakovskaya, 2000). The hydrolysis of polyphosphates to free phosphate resulted in a significant increase in phosphate titre for the wild-type (P <0.05), indicating that the organism produced detectable polyphosphate. There was no significant difference in phosphate titre pre- and post-hydrolysis for the samples isolated from cultures of the mutant, indicating that polyphosphate production had been abolished by inactivation of FTN1472.
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Fig. 3. Organization of the genomes of F. tularensis strain SCHU S4 and F. novicida strain U112 in the region of FTN1472/FTT1564.
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Having been identified in our promoter trapping screen as a gene apparently induced intracellularly, the effect of inactivation of FTT1564 on intracellular growth of Francisella was evaluated in the J774.1 macrophage cell line. Initially, the mutants appeared to be taken up by macrophages similarly to wild-type; however, a growth defect was apparent by 24 h post-infection and significant (P <0.001) by 48 h compared with the wild-type (Fig. 5). The experiment was terminated at 48 h, as Francisella induces destruction of the monolayer after this time. The growth defect was not as marked as that observed following mutation of iglC or its regulator MglA (Baron & Nano, 1998; Golovliov et al., 2003). Thus, similarly to the function of the homologue in the attenuated subsp. holarctica strain LVS (Maier et al., 2007), polyphosphate kinase was shown to be essential for intracellular survival and growth for both subsp. novicida and the highly virulent subsp. tularensis. Inactivation of polyphosphate production has been shown to have a detrimental effect on intracellular survival of other intracellular pathogens such as M. tuberculosis (Sureka et al., 2007) and Salmonella enterica (Kim et al., 2002).
Most mutants of Francisella defective for intracellular survival, such as the iglC mutant, are attenuated, even though they may persist for significant periods (Twine et al., 2005). We wished to determine whether the observed intracellular growth defect correlated with attenuation in the mouse model of disease, as has been indicated by results obtained with a transposon insertion mutant of strain LVS (Maier et al., 2007). BALB/c mice were inoculated by the s.c. route with U112 or U112FTN1472 : : CAM. Retrospective counts determined that mice received 6.9x104 or 6.9x106 c.f.u. of the mutant and 7.25x104 or 7.25x106 c.f.u. of wild-type. Mice were also inoculated by the s.c. route with either SCHU S4 or SCHUS4FTT1564 : : CAM. Retrospective counts determined that mice received 4.5x102 or 4.5x104 c.f.u. of the mutant and 2x102 or 2x104 c.f.u. of wild-type. Both mutants were attenuated such that all mice survived the lower dose challenge, but there were some deaths at the higher challenge levels (Fig. 6a, b). Thus, as was observed following i.p. challenge with the transposon mutant of the attenuated subsp. holarctica strain LVS (Maier et al., 2007), polyphosphate kinase was shown to be essential for virulence for both subsp. novicida and the highly virulent subsp. tularensis.
FTN1472/FTT1564 mutants induce a protective immune response against homologous challenge with subsp. novicida but not subsp. tularensis
To determine whether infection with the attenuated mutants was sufficient to induce a protective immune response, the survivors from the attenuation study were challenged with 100 LD50 of each wild-type strain. The U112FTN1472 : : CAM mutant was able to induce a robust protective immune response against homologous challenge at both dose levels (Table 3). However, mice receiving the lower dose of SCHUS4FTT1564 : : CAM were not protected against wild-type SCHU S4 and there was no delayed time to death. Naïve mice challenged in parallel all died. This may in part reflect the differences in the basis of protection against high- and low-virulence strains, with antibody being sufficient to protect against subsp. novicida, whilst cell-mediated immunity is key to a protective immune response against subsp. tularensis infection (Fulop et al., 2001). The lack of protection at lower immunizing doses of SCHUS4FTT1564 : : CAM may indicate that the mutant is unable to replicate sufficiently to induce a protective immune response. Previously, overattenuation due to a growth defect was reported for a F. tularensis subsp. novicida purA mutant (Quarry et al., 2007). Alternatively, polyphosphate may regulate expression of an antigen that contributes to the protective immune response against tularaemia: loss of polyphosphate kinase has profound effects on cellular composition and morphology (Fraley et al., 2007). Mere persistence by Francisella in vivo has been shown not to correlate with the ability of mutants to induce a protective immune response (Twine et al., 2005) and the correct interaction of the attenuated mutant with the host is key to developing a protective immune response. Further work will be required to elucidate the molecular mechanisms by which FTN1472/FTT1564 contributes to the virulence of Francisella and its intracellular lifestyle.
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ACKNOWLEDGEMENTS
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This work was funded by the UK Ministry of Defence. The authors wish to thank Melanie Duffield and Mark Pallen’s laboratory for bioinformatics assistance, Janine Quarry for technical assistance, Tom Laws for statistical help and Gill Hartley for help with confocal microscopy.
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INTRODUCTION
METHODS
, Unhydrolysed; 
, hydrolysed.
