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Infection and Immunity, May 2007, p. 2591-2602, Vol. 75, No. 5
0019-9567/07/$08.00+0     doi:10.1128/IAI.01789-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

A Defined O-Antigen Polysaccharide Mutant of Francisella tularensis Live Vaccine Strain Has Attenuated Virulence while Retaining Its Protective Capacity{triangledown}

Shite Sebastian,1,2 Simon T. Dillon,3 Jillian G. Lynch,1 LeeAnn T. Blalock,1 Emmy Balon,3 Kristin T. Lee,1 Laurie E. Comstock,2 J. Wayne Conlan,4 Eric J. Rubin,3 Arthur O. Tzianabos,1,2 and Dennis L. Kasper1,2*

Department of Microbiology and Molecular Genetics,1 Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School,2 Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts 02115,3 National Research Council Canada, Institute for Biological Sciences, Ottawa, Ontario KIA OR6, Canada4

Received 9 November 2006/ Returned for modification 3 December 2006/ Accepted 3 February 2007


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ABSTRACT
 
Francisella tularensis, the causative agent of tularemia, has been designated a CDC category A select agent because of its low infective dose (<10 CFU), its ready transmission by aerosol, and its ability to produce severe morbidity and high mortality. The identification and characterization of this organism's virulence determinants will facilitate the development of a safe and effective vaccine. We report that inactivation of the wbtA-encoded dehydratase of the O-antigen polysaccharide (O-PS) locus of the still-unlicensed live vaccine strain of F. tularensis (LVS) results in a mutant (the LVS wbtA mutant) with remarkably attenuated virulence. Western blot analysis and immune electron microscopy studies associate this loss of virulence with a complete lack of surface O-PS expression. A likely mechanism for attenuation is shown to be the transformation from serum resistance in the wild-type strain to serum sensitivity in the mutant. Despite this significant attenuation in virulence, the LVS wbtA mutant remains immunogenic and confers protective immunity on mice against challenge with an otherwise lethal dose of either F. tularensis LVS or a fully virulent clinical isolate of F. tularensis type B. Recognition and characterization of the pivotal role of O-PS in the virulence of this intracellular bacterial pathogen may have broad implications for the creation of a safe and efficacious vaccine.


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INTRODUCTION
 
Francisella tularensis, a pleomorphic gram-negative facultatively intracellular bacterial pathogen, is the etiologic agent of tularemia, a potentially fatal human disease (16). Both of the two major biovars—Francisella tularensis bv. tularensis (type A) and Francisella tularensis bv. holarctica (type B)—are highly virulent and infectious in a wide range of mammalian species, although type A strains tend to be more virulent in higher mammals, including humans (37). The ease with which F. tularensis can be aerosolized and its high degree of infectivity when inhaled have raised concerns about its potential for use in biological warfare or bioterrorism (13). An empirically derived, still-unlicensed vaccine strain of F. tularensis, referred to as live vaccine strain (LVS), was created more than 50 years ago by in vitro passage of a type B clinical isolate and is currently being administered as an investigational new drug to at-risk individuals (33). Unfortunately, more-routine use of F. tularensis LVS as a vaccine is complicated by several issues: (i) the LVS strain is still highly virulent in some animal models of infection; (ii) LVS vaccine has been associated with significant undesirable side effects; (iii) LVS vaccine recipients develop incomplete immunity; and (iv) the molecular basis for the attenuation of LVS is unknown.

In spite of these concerns, LVS does provide an opportunity to identify a key virulence factor(s) that could well prove instrumental in the development of a safer and more effective vaccine. The current understanding of the virulence factors of F. tularensis is rudimentary (16). Several laboratories are attempting to identify these determinants (21, 28, 29). Although surface polysaccharides, including lipopolysaccharide (LPS) and possibly a capsular polysaccharide, have been proposed as important virulence determinants for this organism (16, 34), the lack of strains with defined genetic mutations in the putative polysaccharide loci has hindered studies of the exact role of these molecules in pathogenesis.

The LPS of F. tularensis is similar to that of other gram-negative bacteria in that it consists of lipid A, a core oligosaccharide, and an O-antigen polysaccharide (O-PS) side chain (40). However, unlike the LPSs of most other pathogenic bacteria, the LPS of F. tularensis does not evoke an overt proinflammatory cytokine response and is considered nontoxic (1, 35). The locus encoding the putative O-PS side chain of the LPS has been inferred by in silico analyses of the recently sequenced genome of Francisella tularensis subsp. tularensis Schu S4 (27, 30). These analyses have identified a 17-kb gene cluster that is predicted to code for the enzymes responsible for the biosynthesis of the O-PS repeating unit. Our own in silico analysis of the unannotated genome of F. tularensis LVS has identified an 18-kb locus with homology to the O-PS locus of Schu S4 (Fig. 1A). The putative functions of the gene products in the O-PS loci of both strains (LVS and Schu S4) are consistent with the functions of the enzymes likely to be necessary for the biosynthesis of the known identical structures of the O-PS repeating units of type A and B strains (30). However, the exact role of the gene products in polysaccharide biosynthesis awaits genetic and biochemical confirmation.


Figure 1
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  		FIG. 1. Mutagenesis of F. tularensis LVS. (A) Genetic map of the F. tularensis LVS O-PS locus identified by in silico analysis of the LVS genome. The site of Himar1 Tn insertion in the wbtA gene of the LVS O-PS locus is shown (accession no. DQ353940). (B) Schematic representation of mariner-based mutagenesis of LVS. (Left) Delivery plasmid (pSD) used for mutagenesis of LVS. The Himar1 Tn with ITRs (open arrows) flanking the Kmr gene is represented. (Right) The C9 transposase (C9Tpase) encoded in cis inserts a single copy of the Himar1 Tn at the TA dinucleotide target in the LVS chromosome, resulting in Kmr Himar1 Tn insertion mutants. +, Himar1 insertion in the plus orientation; –, Himar1 insertion in the minus orientation. Shaded square with bent arrow, groEL promoter. (C) Southern hybridization analysis. NcoI-digested chromosomal DNA (5 µg) was electrophoresed, transferred to a Zeta-Probe (Bio-Rad, Hercules, CA) membrane, and probed with the digoxigenin-labeled Tn903 kanamycin resistance gene. Lanes: 1, F. tularensis LVS (negative control); 2, amide ligase (capB); 3, epimerase/dehydratase (wbtA); 4, anthranilate synthase; 5, resistance-nodulation-division efflux protein; 6, ABC transporter; 7, type I restriction (hsdR); 8, oxidoreductase; 9, 2-isopropylmalate synthase; 10, iglD; 11, ferroredoxin. (D) In vitro growth kinetics of the parent strain F. tularensis LVS and the LVS wbtA insertion mutant. The doubling time of the LVS wbtA mutant was compared with that of the parent strain in vitro during growth in modified Mueller-Hinton broth (Difco, Detroit, MI) after inoculation of cultures at an optical density at 620 nm of ~0.05. All growth experiments were performed in triplicate.

We inactivated the wbtA-encoded dehydratase of the O-PS locus of F. tularensis LVS. Studies with the resulting mutant demonstrate the important role of the O-PS and of antibodies to the O-PS of F. tularensis LVS LPS in the pathobiology of this microbe.


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MATERIALS AND METHODS
 
mariner Tn construction and mutagenesis of F. tularensis LVS. The suicide delivery plasmid pSD, designed for the mutagenesis of F. tularensis LVS, contains the following: (i) a Tn903-derived kanamycin open reading frame (ORF) under the control of the 241-bp F. tularensis LVS groEL promoter, (ii) Himar1 inverted terminal repeats (ITRs) flanking the kanamycin resistance gene to generate the mariner minitransposon (Tn), and (iii) the C9 transposase gene. For construction of the pSD delivery plasmid, the 241-bp native groEL promoter was cloned upstream of the Tn903-derived kanamycin ORF by overlap extension PCR (23). Primer pairs SD52 (5'-TCTAGCGGCCGCACTAT ACCCTTCAAGCTTTG-3') and SD38 (5'-CGTTGAATATGGCTCATAACAAT CTTACTCCTTTG-3') were used for amplification of a 241-bp promoter. The promoter elements were then fused to the Tn903-derived kanamycin ORF, which was amplified by PCR with primer pairs SD39 (5'-GGAGTAAGATTGTTATGAGCCATATTCAA CGG-3') and SD40 (5'-GTACGGATTCCAACCCTGAAGCTTGCTTGC-3'). To generate the mariner Tn SD (ITR-241Kan-ITR), the Himar1 ITRs were added to the ends of the gene fusion by cloning the fragments from the previous step into a vector that had the ITRs with cloning sites in between. Primer SD65 (5'-GATTCCGGATAACAGGTTGGC-3') was used for PCR amplification of the transposons and addition of a BspEI site to their ends. In the final step, the mariner Tn SD was cloned into the XmaI site of the Himar1 C9 transposase-expressing plasmid pSC186 (S. Chiang, personal communication). For mutagenesis experiments, the mariner Tn plasmid pSD was introduced into F. tularensis LVS by electroporation as described previously (2), and kanamycin-resistant (Kmr) insertion mutants were selected on cysteine heart agar containing 2% hemoglobin (CHAH) and kanamycin (5 µg/ml). The insertion site in each mutant was identified by genomic sequencing of chromosomal DNA as described previously (25).

Complementation analysis. To obtain the groEL-wbtA fusion product, the 241-bp groEL promoter was amplified (forward primer, 5'-CGGGATCCTATACCCTTCAAGCTTTGAAAAATAA-3'; reverse primer, 5'-GCGTTCTATTATCGTAGAAAGACATAACAATCTTACTCCTTTGTTAAATT-3') and cloned upstream of the F. tularensis LVS WbtA ORF (forward primer, 5'-AATTTAACAAAGGAGTAAGATTGTTATGTCTTTCTACGATAATAGAACGC-3'; reverse primer, 5'-CGGGATCCCTACCCATTCAATCTATGTTCAAATTCCGGAA-3') by overlap extension PCR (underlining denotes the cut site for the BamHI restriction enzyme). The groEL-wbtA fusion product was subsequently cloned into the BamHI site of the Escherichia coli-Francisella shuttle plasmid pHV33 to obtain the complementation plasmid pHV33.wbtA. For complementation studies, the recombinant plasmid pHV33.wbtA was introduced into the mutant strain under standard electroporation conditions (25), and transformants were selected on CHAH supplemented with chloramphenicol (1 µg/ml).

Generation of Francisella mouse antisera. Male BALB/cByJ mice were immunized twice, 2 weeks apart, with F. tularensis LVS (~103 CFU/mouse) or the LVS wbtA mutant (~108 CFU/mouse). All mice were bled 2 weeks after the last immunization, and sera were collected for use in passive serum transfer experiments.

Generation of Francisella rabbit antisera. Whole-organism antisera to the formalin-fixed parent strain LVS ({alpha}-F. tularensis sera) and the LVS wbtA mutant strain were generated in rabbits at Lampire Biological Laboratories (Pipersville, PA) with the standard Classic Line protocol. For preparation of adsorbed antisera, 100 µl of {alpha}-F. tularensis serum was diluted with 10 ml of 1x phosphate-buffered saline (PBS) and mixed with either the F. tularensis parent strain LVS or the LVS wbtA mutant from a 50-ml overnight culture of the parent strain LVS or the LVS wbtA mutant. The suspensions (bacteria plus {alpha}-F. tularensis serum) were incubated on a rotary shaker at 37°C for 1 h, after which the cells were removed by centrifugation. This adsorption procedure was repeated, and the resulting adsorbed antiserum was used in immunoblot analyses.

Immunoelectron microscopy. Electron microscopy studies with CHAH plate-grown bacterial cultures were performed as previously described (24). The primary antibody (monoclonal antibody [MAb] 2033) and the rabbit anti-mouse bridging antibody were used at final dilutions of 1:20 and 1:100, respectively. A secondary antibody tagged with protein A-gold (15-nm-diameter gold particles; Cell Microscopy Center, University Medical Center Utrecht, Utrecht, The Netherlands) was used at a final dilution of 1:65. All images were recorded at a primary magnification of x18,000.

Bactericidal assays. Mid-logarithmic-phase cultivated bacteria were washed and resuspended in sterile 1x PBS prior to use in killing assays. In brief, bacteria (~104 CFU) were incubated with 20% heat-inactivated normal rabbit serum (Lampire) in the presence of either heat-inactivated or active baby rabbit complement (Cedarlane Laboratories, Hornby, Ontario, Canada) at a final dilution of 1:10 in a final volume of 500 µl. The number of CFU per milliliter was determined by plating 10-µl aliquots onto CHAH plates at time zero and incubating for 30 min at 37°C. Serum used as a complement source was heat inactivated at 56°C for 30 min.

Mouse challenge studies. Male BALB/cByJ mice (6 to 8 weeks old; Jackson Laboratory, Bar Harbor, ME) were caged in a microisolator and housed in a pathogen-free environment in the animal facility at Harvard Medical School. Intradermal and intranasal 50% lethal doses (LD50) of F. tularensis LVS for these mice were determined (by the method of Reed and Muench [31a]) to be 106 CFU and 858 CFU, respectively. In a separate study, we determined the virulence potential of the F. tularensis LVS wbtA mutant by challenging BALB/cByJ mice (n = 8) by various routes over a range of doses, as follows: (i) intradermal injection, 3.7 x 103 CFU to 3.7 x 108 CFU (100-µl volume, inoculated into the skin fold of the shaved midbelly); (ii) intranasal instillation, 3.7 x 101 CFU to 3.7 x 107 CFU (50 µl per mouse); and (iii) intraperitoneal injection, 4.0 x 101 CFU to 4.0 x 107 CFU (100-µl volume). Survival was monitored over a 28-day period. All mice that survived the intradermal or intranasal challenge with the LVS wbtA mutant were subsequently challenged with 25 times the LD50 of LVS via the homologous route, and survival was monitored.

Wild-type challenge studies. Specific-pathogen-free female BALB/c mice were purchased from Charles River Laboratories (St. Constant, Quebec, Canada) and were used in experiments starting when they were 8 to 10 weeks old. For both vaccination and challenge, intradermal inocula (50 µl per mouse) were diluted in sterile saline and injected into a fold of skin in the shaved midbelly. For immunization, mice were inoculated once with 5 x 103 CFU of F. tularensis LVS (positive control) or twice (with doses 2 weeks apart) with 1.5 x 108 CFU of the LVS wbtA mutant (39). Challenge with the virulent type B strain FSC 108 (17 CFU) and the type A strain Schu S4 (10 CFU) was performed in a federally certified small-animal level-3 biocontainment facility. For both clinical strains, the intradermal LD50 is <10 CFU and kills mice within 8 days (5).

H&E staining. For histopathologic analysis, reticuloendothelial tissues embedded in paraffin were sectioned and stained with hematoxylin and eosin (H&E). All histopathology sections were evaluated and scored by a single veterinary pathologist in a blinded fashion at Harvard Medical School.

Passive serum transfer studies. Polyclonal antisera (volume, 100 µl) to F. tularensis LVS or the LVS wbtA mutant strain were administered to groups of male BALB/c mice (n = 5) by the intravenous route. The control group received 100 µl of normal mouse serum. All recipient mice were challenged after 24 h with an ordinarily lethal dose of the parent strain LVS (35 times the intradermal LD50), and survival was monitored. In studies using rabbit antisera, 100-µl volumes of rabbit antisera were administered to groups of male BALB/c mice (n = 5) by the intravenous, intracardiac, and intraperitoneal routes. O-PS-adsorbed sera used for passive transfer protection studies were obtained by incubating LVS rabbit antisera with 4 mg of purified LVS O-PS for 1 h at 37°C. Sera (adsorbed and unadsorbed) were transferred via the intraperitoneal route, and recipient mouse groups (n = 6) were challenged 24 h posttransfer with a dose equivalent to 30 times the intradermal LD50.

Statistical analysis. Kaplan-Meier survival analysis and the log rank test to determine P values for the survival curves were performed with GraphPad Prism, version 4.0 (GraphPad Software Inc., San Diego, CA). For dissemination studies, the statistical significances of differences between groups were determined by the one-tailed unpaired t test with the Welch correction using the GraphPad (San Diego, CA) InStat 3 software program.

Nucleotide sequence accession number. The sequence of the F. tularensis LVS wbtA mutant has been deposited in GenBank under accession no. DQ353940.


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RESULTS
 
Mutagenesis of F. tularensis LVS: identification and characterization of polysaccharide mutants. A Himar1-based mariner Tn strategy was employed for the mutagenesis of the F. tularensis LVS chromosome (32). The delivery plasmid does not replicate in the host, and kanamycin resistance is strictly contingent on the transposition of the Himar1 Tn into the LVS chromosome (Fig. 1B). Sequence analyses of representative Kmr insertion mutants indicated no directional bias of Himar1 insertion.

Among the first 10 representative mariner Tn mutants analyzed, we identified one with an insertion in the wbtA gene of the putative O-PS biosynthesis cluster (30) of F. tularensis LVS. As shown in Fig. 1C, a single transposition event of the Himar1 Tn was confirmed by Southern hybridization for this mutant (the F. tularensis LVS wbtA mutant; accession no. DQ353940) and for all the other insertion mutants examined. The stability of the Himar1 Tn insertion in the mutants was also confirmed by chromosomal DNA analyses of mutants before and at the end of a 2-week period of daily passage in the absence of antibiotic selection (data not shown). A quantitative real-time PCR to evaluate the transcriptional status of the sequences immediately downstream of the Tn insertion in the F. tularensis LVS wbtA mutant indicated no down-regulation of these sequences. On the contrary, increases in the transcript levels of the downstream genes were observed (data not shown), consistent with the presence of a native groEL promoter in the Himar1 Tn. In vitro growth studies revealed no obvious growth defect of the mutant strain compared with the parent strain LVS (Fig. 1D).

Surface O-PS expression and biochemical characteristics of the wbtA insertion mutant. We next characterized the wbtA mutant further in order to establish the exact role of O-PS in the pathobiology of F. tularensis LVS. Negative-contrast immune electron microscopy (NC-IEM) studies evaluating surface O-PS expression demonstrated extensive immunogold labeling on the surface of the wild-type strain LVS upon incubation with MAb 2033 (Fig. 2A), whose binding site is mapped to the O-PS of F. tularensis LPS (MAb 2033 product data sheet, Abcam Inc., Cambridge, MA). In contrast, the LVS wbtA mutant displayed no immunogold label (Fig. 2B).


Figure 2
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  		FIG. 2. Lack of O-PS expression in the F. tularensis LVS wbtA mutant strain. (A and B) NC-IEM of the parent strain LVS (A) and the LVS wbtA mutant (B) demonstrating surface immunogold labeling status after incubation with the O-antigen-specific MAb 2033. (C and D) Immunoblot analyses to determine the O-PS expression status of F. tularensis LVS and the LVS wbtA mutant strain. Bacterial cell lysates (10 µl/lane) were electrophoresed on a gradient gel (4 to 20%), transferred to an Immobilon-P membrane, and probed with antibodies. (C) Samples were probed with either {alpha}-F. tularensis serum (rabbit polyclonal antibody to F. tularensis LVS) (panel 1) or a mouse MAb to F. tularensis LPS (panels 2 [MAb 2033; clone FB11] and 3 [MAb 2034; clone T14]). (D) Samples were probed with {alpha}-F. tularensis serum adsorbed with either F. tularensis LVS (panel 1) or the F. tularensis LVS wbtA mutant (panel 2). (E and F) Reconstitution of the O-PS ladder in the complemented strain was examined with MAb 2033 by immunoblot analyses similar to those in panels C and D (E) and by NC-IEM (F). All primary and secondary antibodies were used at final dilutions of 1:3,000 and 1:5,000, respectively, in 1x casein.

Immunoblot analyses of whole-cell lysates revealed a characteristic O-PS ladder pattern for F. tularensis LVS that was completely lost in the LVS wbtA mutant (Fig. 2C). However, distinct bands were visualized in the lane for the LVS wbtA mutant when it was probed with {alpha}-F. tularensis serum (Fig. 2C1). These bands most likely represent the protein antigens of LVS that are obscured when the O-PS ladder is present.

Similar immunoblot analyses conducted with adsorbed serum also indicated a complete loss of O-PS expression in the LVS wbtA mutant (Fig. 2D). No O-PS-specific ladder was detected when cell lysates of the LVS wbtA strain were analyzed with {alpha}-F. tularensis serum adsorbed with either LVS or the LVS wbtA strain (Fig. 2D1 and 2). In analyses with LVS-adsorbed {alpha}-F. tularensis serum, protein antigen bands similar in molecular size to those identified previously were again seen in both the LVS and LVS wbtA mutant lanes (Fig. 2D1). The characteristic laddering polysaccharide pattern was evident when whole-cell lysates of LVS were subjected to immunoblot analyses with LVS wbtA mutant-adsorbed {alpha}-F. tularensis serum (Fig. 2D2).

To ascertain whether the absence of O-PS expression in this strain was due solely to inactivation of the wbtA gene, we complemented the phenotype by introducing an intact copy of the homologous wbtA gene in trans. Immunoblot analyses showed that the ability of the complemented strain to express O-PS was rescued, as evidenced by partial reconstitution of the polysaccharide ladder (Fig. 2E). Surface expression of O-PS in the complemented strain was further confirmed by NC-IEM and enzyme-linked immunosorbent assay inhibition studies (Fig. 2F; also data not shown).

The F. tularensis LVS wbtA mutant strain is attenuated in virulence. Identification of a defined mutation in F. tularensis LVS resulting in the loss of surface O-PS expression allowed us to definitively examine the exact role of this molecule in the virulence and pathogenesis of F. tularensis. As shown in Table 1, all mice challenged intradermally with increasing 10-fold doses of the LVS wbtA mutant survived, even at the highest dose (108 CFU), whereas the LD50 of the parent strain LVS was ~106 CFU. Increasing 10-fold challenges via the intranasal route produced similar survival results with the mutant strain and defined the intranasal LD50 of the parent strain as <103 CFU (Table 1).


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  		TABLE 1. Survival ratios and mean TTD of male BALB/cByJ mice after challenge with F. tularensis LVS and F. tularensis LVS::wbtA mutant strainsa

The attenuation status of the mutant was further scrutinized by challenging mice via the more sensitive intraperitoneal route; the F. tularensis parent strain LVS has a reported lethal infectious dose of ~1 CFU in intraperitoneal challenge studies (14). As shown in Table 1, all mice challenged with the parent strain succumbed to infection, even at the lowest intraperitoneal challenge dose (4 CFU). In contrast, no deaths were recorded up to a dose of 107 CFU among mice challenged intraperitoneally with the LVS wbtA mutant, indicating an LD50 at least 7 log units higher for the LVS wbtA mutant than for the parent strain.

The ability of the F. tularensis LVS wbtA mutant strain to disseminate is severely impaired. We examined the ability of the LVS wbtA mutant to disseminate to and colonize the reticuloendothelial system after either intradermal or intranasal challenge. As shown in Fig. 3A, the bacterial counts in spleens of mice challenged intradermally 4 days previously with the parent strain were ~6.5 log units higher than those in spleens of LVS wbtA mutant-challenged mice. Although the LVS wbtA mutant strain was able to disseminate to a lesser degree than LVS (as evidenced by the detection of the LVS wbtA mutant only at very early times after challenge [data not shown]), the organ-specific bacterial burdens in the kidneys, lungs, or livers of mice challenged intradermally with the LVS wbtA mutant were below the level of detection when examined at day 4 postinoculation. In striking contrast, day 4 organ bacterial counts for mice inoculated intradermally with the parent strain showed at least 8 to 10 log CFU of bacteria per gram of tissue (Fig. 3A). It is noteworthy that the LVS wbtA strain survived in lung tissue sampled 4 days after intranasal challenge of mice (Fig. 3B). However, bacterial counts of the mutant were ~1 log unit lower than bacterial counts in the lungs of mice challenged with the parent strain LVS. Although the mutant replicated in lung tissue after intranasal administration, evidence for a significant limitation on its ability to disseminate to reticuloendothelial tissues was provided by a several-log-unit reduction in splenic counts (Fig. 3B). Increased serum sensitivity has previously been shown to result in a decreased ability to disseminate for other microorganisms (36). In vitro bactericidal assays were performed to examine the mutant's sensitivity to complement-mediated killing. LVS was fully resistant to killing, while the LVS wbtA mutant was highly sensitive (Fig. 3C).


Figure 3
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  		FIG. 3. The F. tularensis LVS wbtA mutant is defective in dissemination and is sensitive to complement. Male BALB/c mice (n = 5) were challenged intradermally (~107 CFU) (A) or intranasally (~104 CFU) (B) with F. tularensis LVS or the LVS wbtA mutant. The number of CFU per gram of tissue was determined 4 days after challenge. To determine the CFU per gram of tissue, reticuloendothelial tissues were harvested, homogenized in 1x PBS supplemented with 2% fetal bovine serum, and plated on CHAH plates. *, P = 0.0024; **, P = 0.0009; ***, P < 0.0001. (C) In a bactericidal assay, bacterial strains were incubated for 30 min at 37°C with heat-inactivated (56°C for 30 min) normal rabbit serum ({Delta}NRS) in the presence of either active complement (+C) or heat-inactivated (56°C for 30 min) complement (+{Delta}C). The number of viable CFU was determined by plating of appropriate dilutions on CHAH plates.

Histopathological assessment studies. The degree of the mutant's impairment in dissemination was also evaluated by histopathologic assessment of liver and spleen tissue. Liver tissue from mice challenged with F. tularensis LVS showed obvious signs of inflammation, with foci of neutrophilic infiltration (Fig. 4F). In stark contrast, liver tissue from mice challenged with the LVS wbtA mutant contained only minor sporadic infiltrations—most likely due to the low numbers of bacteria in these tissues, for which the histology was more consistent with normal than with infected livers (Fig. 4D and E). Spleen histopathology on day 4 showed massive infiltration of neutrophils and macrophages (pyogranuloma) in LVS-challenged mice (Fig. 4C), while normal architecture was evident in spleen tissues from control and LVS wbtA mutant-challenged mice (Fig. 4A and B). The results were similar when an intradermal challenge dose of the LVS wbtA mutant (~108 CFU) was administered (data not shown).


Figure 4
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  		FIG. 4. H&E staining of spleen (A to C) and liver (D to F) tissues 4 days after intradermal challenge with ~107 CFU of the parent strain F. tularensis LVS or the LVS wbtA insertion mutant. The region represented in the inset is demarcated by a black circle in each panel. The asterisk in the panel C inset and the white arrowheads in the panel F inset indicate sites of massive infiltration in spleen and liver tissues after challenge with the parent strain LVS. Minor sporadic infiltrations were observed in the spleen and liver tissues after LVS wbtA challenge (B and E), consistent with those in the normal architecture of spleen and liver tissues (A and D).

Immunization with the F. tularensis LVS wbtA mutant confers protective immunity against subsequent challenge with an otherwise lethal dose of parental LVS and wild-type virulent type B organisms but not against challenge with a type A strain. Having shown that, regardless of the challenge route, the LVS wbtA mutant strain exhibits a markedly reduced ability to cause disease in the murine tularemia model, we next assessed the mutant's protective efficacy. Mice immunized intranasally or intradermally with the LVS wbtA mutant were challenged on day 28 with 25 times the LD50 of F. tularensis LVS via the homologous route. A dose-dependent immunization effect was observed irrespective of the route of immunization, and as few as 104 CFU of the LVS wbtA mutant provided 100% protection against subsequent LVS challenge (Fig. 5A and B).


Figure 5
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  		FIG. 5. Protection of BALB/c mice after immunization with the F. tularensis LVS wbtA mutant. (A and B) Mice (n = 4) were immunized with increasing 10-fold doses of the LVS wbtA mutant by the intranasal route (3.7 x 101 to 3.7 x 107 CFU) (A) or the intradermal route (3.7 x 103 to 3.7 x 108 CFU) (B) and were challenged at day 28 via the homologous route with a lethal dose of F. tularensis LVS (25 times the respective LD50). Survival was monitored over a 28-day period. For clarity, only results for selected immunization doses are shown. (C and D) For wild-type challenge studies, mice (n = 5) immunized with F. tularensis LVS or the LVS wbtA mutant were challenged intradermally 8 weeks after initial immunization with a lethal dose of either the fully virulent type B strain FSC 108 (17 CFU) (C) or the fully virulent type A strain Schu S4 (10 CFU) (D) of F. tularensis.

We next assessed whether immunization with the F. tularensis LVS wbtA mutant protected mice against an otherwise lethal intradermal challenge with fully virulent type B or type A F. tularensis. For wild-type challenge studies, mice were inoculated once with 5 x 103 CFU of F. tularensis LVS (positive control) or twice (with doses 2 weeks apart) with 1.5 x 108 CFU of the LVS wbtA mutant. Considering the significant impairment of the mutant strain in dissemination and its inability to cause disease, we reasoned that a prime-and-boost regimen of the LVS wbtA strain might elicit efficient protective immunity without causing illness, especially against challenge with a highly virulent wild-type strain. As expected from earlier work, mice immunized with LVS showed overt signs of infection (piloerection; between 4 and 7 days) after vaccination (39). In stark contrast, mice immunized with 100,000-fold-greater doses of the LVS wbtA strain showed no overt signs of illness, although the animals did develop transient dermal necrosis at the injection site. As shown in Fig. 5C, after a lethal intradermal challenge (17 CFU) with the fully virulent F. tularensis type B strain FSC 108, all naïve mice succumbed. When the same challenge was administered to mice immunized with the LVS wbtA mutant, all animals initially (days 4 to 12) showed some signs of infection, from which they recovered fully (Fig. 5C). On day 21 postchallenge, all surviving mice were sacrificed, and the numbers of FSC 108 bacteria in their spleens were determined. At this time the spleens from LVS-immunized mice appeared normal, whereas all the mice immunized with the LVS wbtA mutant strain displayed splenomegaly and had a CFU of 4,796 + 1,457 (mean + standard error of the mean). In sharp contrast, upon intradermal challenge with the virulent type A strain Schu S4, all mice immunized with the LVS wbtA mutant showed obvious signs of infection and succumbed (Fig. 5D). As expected, all mice immunized with the parental strain LVS survived this low but ordinarily lethal intradermal challenge with the type A strain (Fig. 5D).

Antibodies to the O-PS of F. tularensis LVS play a pivotal role in humoral immunity. The isolation of a defined O-PS mutant of F. tularensis LVS afforded us a unique opportunity to examine the role, if any, of serum antibodies to LVS protein antigens in humoral immunity. Groups of mice were immunized twice, 2 weeks apart, with either LVS (~103 CFU/mouse) or the LVS wbtA mutant (~108 CFU/mouse), and serum was obtained 2 weeks after the last immunization. The titer of immunoglobulin G (IgG) antibodies directed toward the surface-exposed protein antigens in anti-LVS wbtA antisera was determined to be 8,000 + 1,849, fourfold higher than the titer for LVS-immunized mice (2,000 + 120). Further analysis of the antibodies showed that the IgG2a subclass predominated among the IgG subtypes analyzed in both immunized groups. Groups of naïve mice received (by the intravenous route) 100 µl of pooled polyclonal immune mouse serum (IMS) generated either to the LVS wbtA mutant or to the parental strain LVS. All recipients were challenged after 24 h with 35 times the intradermal LD50 of the parental strain LVS. As expected, all recipients of LVS polyclonal IMS survived this otherwise lethal LVS challenge (Fig. 6). However, all mice that had received the LVS wbtA immune serum succumbed to infection, as did control mice that had received normal mouse serum (Fig. 6).


Figure 6
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  		FIG. 6. Passive serum transfer studies. Polyclonal IMS raised individually against either the parent strain F. tularensis LVS or the LVS wbtA mutant was used. Serum from naïve mice (NMS) served as a control. For protection studies, a 100-µl volume of pooled IMS or NMS was transfused intravenously into naïve BALB/c mice. Recipient mice were intradermally challenged 24 h later with a lethal dose of LVS (35 times the intradermal LD50), and survival was monitored. The serum transfer recipient groups included those receiving LVS IMS (n = 5), LVS wbtA IMS (n = 6), and NMS (n = 5). ***, P = 0.0015 for the LVS serum transfer group versus the group receiving NMS.

The inability of F. tularensis LVS wbtA antisera to protect against a lethal F. tularensis LVS challenge was also confirmed with rabbit polyclonal antisera generated against the LVS and LVS wbtA strains by using the manufacturer's standard Classic Line protocol (Lampire Biological Laboratories, Pipersville, PA). The IgG titers against the protein antigens were determined to be 32,000 + 290 and 64,000 + 665 in LVS and LVS wbtA rabbit antisera, respectively. For passive transfer studies, 100 µl of undiluted serum was transferred to mice in different groups via the intraperitoneal, intravenous, or intracardiac route. All recipient mice were challenged with 30 times the intradermal LD50 after 24 h, and survival was monitored. Regardless of the route of passive antibody transfer, all mice receiving the polyclonal serum generated against the LVS wbtA strain succumbed to infection. In contrast, all mice receiving LVS polyclonal sera survived this challenge dose. An additional study using a 1:2 dilution of the LVS wbtA antiserum was done and ruled out the possibility that these results were due to a prozone-like phenomenon.

The fact that antibodies to LVS protected against LVS challenge whereas antibodies to the O-PS-deficient LVS wbtA mutant did not protect against the same challenge suggested an important protective role for antibodies to the O-PS in LVS antiserum. The pivotal role of antibodies directed toward LVS O-PS was confirmed by passive transfer protection studies using LVS rabbit antisera adsorbed with O-PS. All mice receiving O-PS-adsorbed LVS antisera succumbed to infection by day 7, as did the untreated controls, while mice receiving unadsorbed LVS immune sera survived this lethal challenge with LVS.


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DISCUSSION
 
The wbtA-encoded enzyme family is present in many diverse bacterial polysaccharide biosynthesis loci and is involved in the synthesis of complex nucleotide-linked monosaccharides from less complex nucleotide sugars. The WbtA protein displays similarities to several members of the epimerase/dehydratase family, most strongly resembling PglF of Campylobacter jejuni (17), WbpM of Pseudomonas spp. (3, 4), and YveM of Bacillus subtilis (26). The WbpM protein of Pseudomonas aeruginosa, which is the best-characterized member of the dehydratase subfamily (3, 12), is highly conserved and essential in all Pseudomonas serotypes that contain D-QuiNAc or its derivatives in the O-antigen repeating unit (3). Here we report that a defined mutation in the wbtA-encoded epimerase/dehydratase of the F. tularensis LVS O-PS locus results in a strain that, although severely attenuated in virulence, induces effective protective immunity against subsequent lethal challenge in a murine tularemia model.

What factors account for the high degree of virulence attenuation in the F. tularensis LVS wbtA strain? Early in F. tularensis infection, a transient bacteremic phase facilitates the pathogen's efficient dissemination to and colonization of the reticuloendothelial system (16). In pathogenic microorganisms, the surface polysaccharides, including O-PS and/or capsular polysaccharide, are considered key virulence determinants that can "cloak" the surface of the organism and confer resistance to host-mediated complement lysis during this early stage of infection (16). It has been proposed that F. tularensis LVS evades complement lysis by means of a bacterial capsule (34); however, neither the genetic loci nor the structural identity of the putative capsule has yet been elucidated. Dissemination and histopathology studies of the LVS wbtA mutant clearly demonstrate that although the lack of O-PS in this mutant did not completely abolish its ability to disseminate, it significantly impaired its ability to disseminate efficiently to the vital organs (presumably due to its increased sensitivity to complement lysis), a defect that significantly impaired the organism's disease-producing capacity. The increased sensitivity of the LVS wbtA mutant to complement lysis was also confirmed by the inability to culture the mutant strain from blood more than 1 day after challenge, in contrast to the parent strain, which was recovered in large numbers 4 days after challenge (data not shown). Our findings are consistent with recent observations of increased serum sensitivity of an O-PS-negative mutant of LVS as reported by Raynaud et al. (31) and with previous observations of strains of Francisella tularensis subsp. novicida with LPS biosynthesis gene mutations (10) and of a gray opacity variant of LVS that lacks surface O-PS expression; however, the mutational event(s) underlying the opacity phenotype in the latter two studies remains unclear and could involve alterations in the expression of virulence determinants in addition to O-PS (22). The present study does not address the nature or the role of capsule, if any, in LVS-mediated serum resistance but does provide unequivocal evidence for a major role of O-PS in conferring resistance to serum.

Our demonstration that immunization with the F. tularensis LVS wbtA mutant afforded protection against a low but ordinarily lethal intradermal challenge dose of either F. tularensis LVS or virulent type B organisms but not against a low-dose intradermal type A challenge may reflect the differences in pathobiology and virulence among these strains. These differences may, in turn, obscure the nature of the protective immunity required to combat infection by the different strains. The complexity of the cellular and humoral immunity required to protect against low- and high-virulence strains of F. tularensis is well described in the literature. F. tularensis is an intracellular pathogen, and it is assumed that long-term protective immunity to this organism relies mainly on T-cell-mediated events. Studies by several laboratories have confirmed the important roles of CD4+, CD8+, and double-negative T-cell subsets in the resolution and control of F. tularensis infection (7, 9, 11, 15, 41). Our studies delineating the nature of protective immunity following immunization with the LVS wbtA strain further substantiate a critical role for cellular immunity in protection against this intracellular pathogen. Nonetheless, it is important to point out that T-cell-independent antibody-mediated resistance to F. tularensis infection has also been demonstrated. In brief, LVS LPS immunization and passive transfer of the resulting antibodies have been shown to effectively control infection after an otherwise lethal dose of LVS but not of fully virulent type A F. tularensis (18-20). The role of humoral immunity in protection has been further substantiated by studies demonstrating the ability of mice immunized with either LVS LPS or a subunit (bovine serum albumin-O-PS) vaccine to survive subsequent challenge with an ordinarily lethal dose of LVS or a fully virulent type B strain but not with a virulent type A strain of F. tularensis (6, 8).

Passive transfer experiments performed in the present study, using IMS and rabbit sera raised to the F. tularensis parental strain LVS and the LVS wbtA mutant, indicate that protective antibodies directed specifically toward the O-PS of LVS LPS are an important component of humoral immunity. However, antibodies elicited against other antigens (including surface proteins) by LVS wbtA immunization do not appear to be protective against a lethal LVS intradermal challenge under the conditions examined in this study. In general, our findings are in accord with previous reports (20) of the important role of antibodies to F. tularensis LPS in protection against the low-virulence strain LVS.

Although this and other studies point to an imperative role for antibodies in controlling infection with low-virulence F. tularensis LVS or a fully virulent type B strain, active immunization with the F. tularensis LVS wbtA mutant (which completely lacks O-PS) effectively controlled infection after a low but ordinarily lethal intradermal challenge with the parental strain LVS or a fully virulent type B clinical isolate. On the other hand, active immunization with LVS—but not with the LVS wbtA mutant—protected mice against a low but otherwise lethal intradermal challenge with a fully virulent type A strain. Therefore, active immunization with the LVS wbtA mutant obviously elicited additional protective immune responses that were independent of antibodies to O-PS and that controlled infection after challenge with LVS or virulent type B organisms but not after challenge with a virulent type A strain. Our finding that immunization with the LVS wbtA strain (an O-PS-negative strain) does not confer protective immunity against a virulent type A strain is consistent with the recent findings of Thomas et al., who have also reported the inability of a similar O-PS-negative mutant strain to protect against subsequent type A challenge (38).

As noted previously, subunit (bovine serum albumin-O-PS) vaccination induces high titers of antibody to O-PS, delaying the mean time to death of mice challenged with a fully virulent type A strain of F. tularensis (6). The major difference between the two immunizing strains used in the present study lies in the complete lack of surface O-PS expression in the mutant strain. In light of this study, it appears likely that the protection observed after immunization with F. tularensis LVS was at least partly due to the presence of antibodies to O-PS, which delayed the progression of disease due to the virulent type A strain, thereby allowing sufficient time for reactivation of the critically important cellular immune defenses generated by the vaccine. Conversely, the lack of O-PS-specific antibodies in LVS wbtA-immunized mice would result in unrestricted growth and dissemination of the type A strain upon intradermal challenge with type A bacteria. Although protection was observed in this study, it remains to be seen whether the LVS wbtA mutant confers protection against a more relevant intranasal or aerosol challenge with either type A or type B organisms. Further studies are warranted to address this issue and to elucidate the mechanism underlying the protective immunity induced by immunization with the LVS wbtA mutant.

During the preparation of this article, a concurrent independent study by Raynaud et al., detailing the role of the F. tularensis LVS O-PS molecule in virulence, was in press as a Note (31). Although our study and the study by Raynaud et al. both indicate an important role for O-PS in virulence and pathogenesis, there are significant differences between these two publications. Our study is a significant expansion of the information provided in the recent publication by Raynaud et al. (31) in the following respects. (i) Raynaud et al. demonstrated that insertion of EZ::Tn in the wbtA gene completely abolished the expression of the downstream genes. In our study, expression of the downstream genes was intact, as demonstrated by quantitative real-time PCR analyses. Furthermore, we were able to partially complement the wbtA mutant phenotype. (ii) Raynaud et al. examined only the attenuation and dissemination status of the O-PS mutant following challenge via the intraperitoneal route. We have examined the degree of attenuation of the F. tularensis LVS wbtA mutant strain and its ability to disseminate via the more clinically relevant intradermal and intranasal routes of infection. (iii) We have assessed the highly avirulent nature of the LVS wbtA mutant by pathological assessment (H&E staining of the reticuloendothelial tissues). (iv) Our study demonstrates that protective immunity exists following immunization with the LVS wbtA strain via both the intradermal and intranasal routes as opposed to only the intraperitoneal route. (v) In addition to lethal LVS challenge (the only challenge strain used by Raynaud et al.), we have demonstrated that protective immunity raised following immunization with the LVS wbtA strain exists against a lethal dose of a highly virulent type B strain (FSC 108) but not against a type A strain (Schu S4) of F. tularensis. (vi) Our studies with wild-type type A challenge point toward the previously unappreciated role of antibodies directed to the O-PS in protection against F. tularensis type A strains, because in contrast to immunization with the LVS strain, the LVS wbtA (O-PS-negative) mutant failed to protect. Based on these findings, it is tempting to speculate that O-PS antibodies might play a critical role in protection against a type A challenge. Further studies are warranted to substantiate these initial findings and are currently under way.

In summary, we describe a defined mutation in F. tularensis LVS that markedly reduces virulence. The wbtA mutant is devoid of the O-PS side chain of LPS and does not efficiently disseminate in the bloodstream of animals, presumably because of mutation-associated acquisition of sensitivity to serum. These studies unequivocally demonstrate a pivotal role of the wbtA gene in the virulence of F. tularensis LVS.


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ACKNOWLEDGMENTS
 
We thank Karen Elkins (U.S. Food and Drug Administration, Bethesda, MD) for providing us with F. tularensis LVS and expert advice. We also thank R. T. Bronson, D. H. Boyd, and M. R. Chase for expert contributions and Maria Ericsson, John E. Warner, and R. J. Panzo for technical assistance.

This study was supported by NERCE grant R01-AI47484 to D.L.K., by grant AID P01AI56296 to Laurie Glimcher, and by grant AI48474 to J.W.C.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115. Phone: (617) 525-2280. Fax: (617) 525-0080. E-mail: dennis_kasper{at}hms.harvard.edu Back

{triangledown} Published ahead of print on 12 February 2007. Back

Editor: A. Casadevall


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Infection and Immunity, May 2007, p. 2591-2602, Vol. 75, No. 5
0019-9567/07/$08.00+0     doi:10.1128/IAI.01789-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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