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1 Center for Molecular Medicine and Infectious Diseases, Virginia–Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
2 Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA
3 Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI, USA
Correspondence
Thomas J. Inzana
tinzana{at}vt.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this work.
Present address: The Institute for Genomic Research, Rockville, MD, USA.
Present address: College of Pharmacy, University of Southern Nevada, Henderson, NV, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Penn, 2005; Sjöstedt, 2005; Timoney et al., 1988; Titball & Sjöstedt, 2002). F. tularensis can be transmitted to humans by direct contact with infected animals, ingestion of contaminated food or water, bites from infected vectors such as ticks and mosquitoes, and inhalation of infectious aerosols or dust (Hopla & Hopla, 1994; Penn, 2005; Titball & Sjostedt, 2002). There are four subspecies of F. tularensis: subsp. tularensis (type A), subsp. holarctica (type B), subsp. mediasiatica and subsp. novicida (Sjöstedt, 2005). However, the International Systematics Committee recognizes Francisella novicida as a separate species (http://www.bacterio.cict.fr/aldl.html). F. tularensis subsp. tularensis is the most virulent subspecies for humans, with an infectious dose of as few as 10 cells (Dennis et al., 2001). Patients may present with several clinical forms of tularöemia, the most common being ulceroglandular tularöemia; other forms include oculoglandular, oropharyngeal, intestinal, pneumonic and typhoidal tularöemia. Pneumonic tularöemia has a mortality rate of up to 30 % in the absence of antibiotics (Ellis et al., 2002; Penn, 2005). Based on these factors, F. tularensis has been classified as a category A select agent by the National Institutes of Health and the Centers for Disease Control (Darling et al., 2002; Dennis et al., 2001).
F. tularensis subsp. tularensis is predominately found in North America, and genetically falls into two subtypes: type A East and type A West (Staples et al., 2006). In contrast, subsp. holarctica is more widely distributed and has been isolated in Europe, Asia and North America. Type B strains are associated with morbidity and a somewhat lower mortality rate in humans that type A East strains (Staples et al., 2006). However, F. novicida is considerably less virulent in humans, and is rarely associated with severe disease, although it is highly virulent in mice (Ellis et al., 2002; Hopla & Hopla, 1994; Titball & Sjöstedt, 2002).
The factors that are associated with virulence in F. tularensis are not well known. Bacterial surface components that contribute to the disease process, or are important for host immunoprotection, have not been clearly identified. Most of the work on F. tularensis surface components has focused on the LPS. The LPS of F. tularensis types A and B is unusual in that the O antigen consists entirely of dideoxyglycoses, the core oligosaccharide contains mannose in place of heptose (Vinogradov et al., 2002, 1991), and lipid A of the live vaccine strain (LVS) is tetraacylated and lacks phosphate (Vinogradov et al., 2002), while lipid A from a virulent type B isolate also contains a phosphate-linked galactosamine (Phillips et al., 2004). Furthermore, the LPS does not signal through TLR4, is not an agonist for TLR4, and does not induce an inflammatory response (Chen et al., 2005; Cole et al., 2006; Hajjar et al., 2006), which is probably due to the atypical structure of lipid A. However, apart from failing to incite an inflammatory response by the host, the role of the LPS in virulence and immunoprotection is unclear.
The genomes of at least five strains of F. tularensis types A and B and F. novicida have recently been sequenced (available at the National Center for Biotechnology Information), and the genome of type A strain Schu S4 has been annotated (Larsson et al., 2005). However, little information is available regarding genomic differences between the highly virulent F. tularensis and the much less virulent F. novicida. In order to identify novel genes that may be responsible for virulence in F. tularensis, we previously used suppression subtractive hybridization (SSH) with subsp. holarctica LVS as the tester and F. novicida strain U112 as the driver (Ahmed & Inzana, 2004). Of 76 LVS-specific genes identified, several were found in the wbt O-antigen locus of LVS that were absent in F. novicida. One LVS-specific gene, which encoded a galactosyl transferase, was selected for mutagenesis. The mutant was devoid of O antigen, was more resistant to the bactericidal effects of sodium deoxycholate than its parental strain, was serum susceptible and attenuated in mice, and provided partial protection against an intraperitoneal (IP) high-dose challenge with the parental strain. The significance of O antigen in F. tularensis virulence and induction of host protection is discussed.
This work was presented, in part, at the mid-Atlantic Microbial Pathogenesis meeting (Wintergreen Conference Center, Charlottesville, VA, February 2004).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. F. tularensis LVS was obtained as a vaccine vial from Dr May Chu, Centers for Disease Control (April, 2002), was subcultured on chocolate agar, and the cells were suspended in sterile skimmed milk and stored at –80 °C.
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and Mach1 T1R (Invitrogen) cells were grown at 37 °C in Luria–Bertani (LB) medium (Difco Laboratories) containing, as appropriate, 100 µg ampicillin (Amp) ml–1, 10 µg chloramphenicol (Cm) ml–1 or 50 µg kanamycin (Km) ml–1 for selection of recombinant strains. F. tularensis and F. novicida strains were grown in Difco Brain Heart Infusion (BHI) broth (Becton Dickinson) supplemented with 0.1 % L-cysteine hydrochloride monohydrate (Sigma) (BHIC) at 37 °C. For culture on agar plates, 5 % (v/v) sheep blood was added to BHIC agar (BHIBC), and the cultures were incubated at 37 °C in 5 % CO2, unless otherwise stated. Actinobacillus pleuropneumoniae was grown in supplemented BHI broth or agar, as described previously (Ward et al., 1998).
DNA manipulation.
Plasmid isolation, DNA restriction endonuclease digestion, ligation and transformation procedures were carried out using standard protocols (Sambrook et al., 1989). Restriction enzymes and T4 DNA ligase were obtained from New England BioLabs. QIAprep Spin Miniprep and QIAquick gel extraction kits (Qiagen) were used to prepare E. coli plasmid DNA. A PUREGENE DNA isolation kit (Gentra Systems) was used to purify genomic DNA from F. tularensis.
Isolation of LVS mutants.
Plasmid pPV, containing a sacB–CmR cassette that confers sucrose sensitivity and resistance to chloramphenicol on Gram-negative cells (Golovliov et al., 2003), was used to mutagenize SSH-identified clone 2-042 (Ahmed & Inzana, 2004), which was later determined to be wbtB from the O-antigen gene cluster (Prior et al., 2003). A 1500 bp region upstream and a 1500 bp region downstream (Table 1
) of wbtB were amplified by PCR using primers FA6-7-NF and FA6-7-NR, and FA6-7-CF and FA6-7-CR, respectively. A typical PCR reaction consisted of 1x PCR HIFI SuperMix (Invitrogen), 0.02 µg genomic DNA as template, and 0.4 µM of each oligonucleotide primer in 50 µl of reaction mixture. The PCR cycling parameters used were 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 52 °C for 30 s, and 68 °C for 2 min, and an additional extension for 5 min at 68 °C. The 5' primers included a SalI restriction site and the 3' primers incorporated either a BamHI restriction site or a PstI restriction site. The upstream and downstream PCR products were digested with SalI/BamHI and SalI/PstI, respectively, and separately cloned into pBluescriptKS+ (Stratagene). The two recombinant clones were digested with SalI/BamHI and both the upstream and downstream insert fragments were ligated simultaneously into SalI-digested pPV, resulting in plasmid pFA501, which was electroporated into E. coli S17-1. Conjugation of LVS with E. coli S17-1(pFA501) was carried out as described elsewhere (Golovliov et al., 2003). After 4 days of incubation, the colonies were transferred to BHIBC agar containing 100 µg polymyxin B ml–1 (to inhibit the donor strain) and 10 µg Cm ml–1. Cm-resistant and polymyxin B-resistant colonies were screened for the presence of the pPV-encoded Amp-resistance gene, which was confirmed by PCR, indicating that pFA501 had integrated into the genome, since the plasmid cannot replicate in the LVS. The Cm-resistant colonies were subcultured onto medium containing 5 % sucrose to select for a second recombination event. Sucrose-resistant, Cm-sensitive colonies were isolated to identify clones that had deleted the plasmid from the genome and presumably also the wild-type wbtB gene. Sucrose-resistant colonies were screened for diminished iridescence (indicating a loss of surface carbohydrate) under incandescent light on BHIC agar, followed by further selection of the non-iridescent colonies on BHIC agar containing 75 mg Congo red l–1. Screening for dark-red colonies on Congo red agar was used because colonies of F. novicida exhibited the dark-red phenotype, whereas colonies of LVS (and type A) were salmon to light pink, indicating surface-component-inhibited binding of Congo red by neutral carbohydrate structures (data not shown). Two colonies that were darker red than the LVS parent were isolated, and PCR (described above) using primers FA5-8NF1 and FA5-8CR1 and Southern blotting were performed to examine the deletion of wbtB.
Plasmid construction and wbtI complementation.
A 2.0 kb DNA fragment containing wbtI from the LVS was amplified by PCR using primers F-wbtI-EcoRI and R-wbtI-NheI, as described above. The resulting fragment was cloned into pCR2.1-TOPO, digested with EcoRI and NheI, gel-purified, and subcloned into the corresponding sites in pFNLTP6 (Maier et al., 2004), resulting in pTZ817. The pTZ817 plasmid was then digested with KpnI and ligated with a KpnI-digested fragment carrying the F. tularensis groE promoter, resulting in pTZ819, which expressed wbtI from the groE promoter. All insert sequences were confirmed by DNA sequencing. Plasmids pTZ817 and pTZ819 were then electroporated into F. tularensis WbtIG191V. The bacteria were grown to a density of about 110 Klett units (correlating to about 109 c.f.u. ml–1), washed three times with 0.5 M sucrose, and suspended in 10 ml 0.5 M sucrose. The plasmid DNA was then electroporated into the bacteria in a 0.1 cm cuvette using an Electro Cell manipulator ECM630 (BTX), as described previously (Maier et al., 2004). Immediately after electroporation, the cells were suspended into 1 ml of TSB without cysteine and incubated with shaking at 37 °C for 4 h before selection on BHIBC agar containing 20 µg kanamycin ml–1.
Sequence alignments and computational structural modelling.
DNA and protein sequences were aligned with CLUSTAL W (Thompson et al., 1994). The tertiary structures of the wild-type protein WbtI, and mutant proteins WbtIS187Y and WbtIG191V were modelled as previously described (Li et al., 2005) to predict the conformational changes caused by residue substitution. Helicobacter pylori aminotransferase Psec (PDB_ID=2FNU, Chain_ID=2FNU_B) with 375 amino acids from the Protein Data Bank (PDB) was used as template for WbtI on the 3D-JIGSAW version 2.0 comparative modelling server (Bates & Sternberg, 1999) (http://www.bmm.icnet.uk/
3djigsaw/). The coordinates in PDB formats for 3-D structures of WbtI were constructed and displayed with RasMol version 2.7.3 (Sayle & Milner-White, 1995).
Extraction of LPS.
LPS was purified by aqueous phenol extraction and ultracentrifugation from killed cells, as described by Vinogradov et al. (1995), with modifications. The bacteria were scraped off BHIBC agar into PBS, and following phenol extraction, 4 vols distilled water was added and the mixture dialysed with tap water until no phenol odour remained. Sodium acetate (pH 7.0) was added to a final concentration of 30 mM, 2 µg DNase I ml–1 from bovine pancreas (Sigma-Aldrich) was added and the mixture was incubated for 2 h at 37 °C, followed by an additional 2 h incubation with 2 µg RNase I ml–1 from bovine pancreas (Amersham Pharmacia Biotech). Proteinase K (Sigma-Aldrich) was then added at 20 µg ml–1 and the mixture was incubated at 37 °C for 2 h. Insoluble material was removed by centrifugation at 10 000 g at 4 °C for 10 min. The LPS was sedimented by ultracentrifugation overnight at 100 000 g at 4 °C, and resuspended in water. The low-speed/high-speed differential centrifugation process was repeated (the low speed was changed to 3000 g for 15 min) until the A260 and A280 of the supernatant were less than 0.02, and the LPS was lyophilized.
PAGE and Western blotting.
The LPS electrophoretic profile was resolved by SDS-PAGE on Novex 16 % Pre-Cast Tricine Gels (Invitrogen) and Emerald Q fluorescence staining (Molecular Probes), as described by the manufacturer. Western blotting was carried out using a Trans-Blot SD semi-dry transfer cell (Bio-Rad), and the blots were developed with rabbit polyclonal antiserum to subsp. holarctica LVS (Inzana et al., 2004), rabbit polyclonal antiserum to F. novicida U112 (1 : 1000 dilution each) or murine mAb (Chemicon International) to F. tularensis O antigen at 1 : 500 dilution. Anti-rabbit IgG or anti-murine IgG coupled to horseradish peroxidase (HRP; Jackson ImmunoResearch Laboratories) were used as secondary conjugates at 1 : 2000 dilution, and colour was developed with 4-chloro-1-naphthol (Bio-Rad). Rabbit polyclonal antiserum to strain U112 (killed by irradiation) was raised in a New Zealand White rabbit, as previously described (Inzana et al., 2004).
LPS composition and structure.
The glycosyl composition of LPS was determined by combined GC/MS of the per-O-trimethylsilyl derivatives of the monosaccharide methyl glycosides, which were produced by acidic methanolysis (Merkle & Poppe, 1994; York et al., 1985). The samples were then per-O-trimethylsilylated by treatment with Tri-Sil (Pierce Chemical) at 80 °C for 30 min. GC/MS analysis of the per-O-trimethylsilyl methyl glycosides was performed on an HP 5890 GC interfaced to a 5970 MSD, using an All Tech EC-1 fused silica capillary column (30 mx0.25 mm internal diameter).
For structural analysis LPS was hydrolysed 4 h in 0.1 M sodium acetate, pH 4.5, at 100 °C. Lipid A was extracted using 2.5 vols chloroform : methanol (2 : 1), and the remaining aqueous phase was desalted on Dowex 50WX8 and lyophilized. The resultant oligosaccharide core was suspended in dry DMSO. The sample was then permethylated by the method of Ciucanu & Kerek (1984), suspended in 5 mM EDTA in methanol : water (1 : 1), and desalted by adding a few microlitres to a few grains of Dowex 50WX8, previously converted into the ammonium salt. The desalted sample was deposited, along with an equal volume of 20 mM dibasic ammonium citrate, onto a thin layer of matrix, whose components were 200 mg trihydroxyacetophenone ml–1 in methanol, and 15 mg nitrocellulose ml–1 in acetone : 2-propanol (1 : 1, v/v), mixed in a 4 : 1 (v/v) ratio (Sturiale et al., 2005). MALDI-MS analysis was then performed using an Applied Biosystems 4700 mass spectrometer operating in the positive ion mode.
Sensitivity to sodium deoxycholate.
The susceptibility of the LVS, mutant strain WbtIG191V and complemented strain WbtIG191V : : pFNLTP/wbtI to the bactericidal activity of sodium deoxycholate was evaluated as described elsewhere (Cowley et al., 2000), with modifications. An equal volume of sodium deoxycholate (Sigma) in PBS was added to wells containing 5x105 c.f.u. of each strain in 100 µl to final concentrations (w/v) of 0, 0.01, 0.1, 1 or 10 %. After 45 min incubation at 37 °C, 20 µl of a 1 : 100 dilution of the mixtures was spread onto BHIBC agar, and bacterial viability was determined after up to 5 days incubation.
Serum bactericidal assay.
The bactericidal activity of 0–40 % pre-colostral calf serum (PCS, which contains no antibodies) for the LVS, mutant WbtIG191V and WbtIG191V : : pFNLTP/wbtI was determined as previously described (Inzana & Anderson, 1985). Control tubes contained serum-sensitive A. pleuropneumoniae J45-100 in place of F. tularensis, or heat-inactivated PCS.
Survival in J774A.1 cells.
Intracellular growth of the LVS, mutant WbtIG191V and the complemented mutant WbtIG191V : : pFNLTP/wbtI was monitored in the murine macrophage-like cell line J774A.1 (American Type Culture Collection) by modification of published methods (Cowley & Elkins, 2003). The number of bacteria added was confirmed by viable plate counting, and used at a m.o.i. of 50 : 1 (bacteria : macrophages). After 2 h incubation of F. tularensis with J774A.1 cells at 37 °C in 5 % CO2, extracellular bacteria were removed by washing the cells with PBS, and the medium was replaced with 1 ml complete Dulbeccos's Modified Eagle Medium (DMEM) plus 50 µg gentamicin ml–1 to eliminate extracellular bacteria. After 45 min incubation, the cells were washed three times with PBS, followed by the addition of complete DMEM without antibiotics. The cells were incubated at 37 °C in 5 % CO2 for 72 h post-infection. The J774A.1 cells were washed in PBS and lysed by exposure to water for 3 min, and serial dilutions of the lysate were plated on BHIBC agar to determine the number of intracellular bacteria at 0 h and at the indicated time points.
Virulence and immunoprotection studies in mice.
To assess virulence, groups of five BALB/c mice 6–8 weeks old (Jackson Laboratory) were challenged IP with various doses of exponential-phase LVS (200, 600 or 2000 c.f.u. per mouse), mutant WbtIG191V (103, 104, 5x104, 1.4x106 or 2.8x107 c.f.u. per mouse) or complemented mutant WbtIG191V : : pFNLTP/wbtI (104 c.f.u. per mouse) in 100 µl PBS. IP inoculations were used to assess virulence because the LVS is most virulent to mice by this route (Fortier et al., 1991), and therefore this route is the greatest test for attenuation. For tissue-clearance studies, 3–5 mice were inoculated intranasally (IN) with 107 c.f.u. per mouse of LVS, WbtIG191V or WbtIG191V : : pFNLTP/wbtI in 20 µl PBS. IN challenge was also used because it is a natural but still severe challenge route (Conlan et al., 2003), and is a measure of the capability of a strain to disseminate. Mice were anaesthetized with mixed oxygen/isofluorane gas prior to inoculation. All inoculation doses were confirmed by viable plate counting on BHIBC agar. Animals exposed to the LVS or mutant strains were maintained and cared for in an accredited ABSL-2 facility. In some cases, mice were euthanized with excess CO2 at 2, 4, 8 or 16 days post-challenge, and bacterial numbers in the spleen, liver and lung were determined following tissue homogenization, serial dilution and viable plate counting.
Mice were immunized either intradermally (ID) or IP followed by IP challenge, which was used because the LD50 of LVS for mice is lowest by the IP route (Fortier et al., 1991). For ID immunization, groups of five BALB/c mice each were inoculated with 100 µl LVS (104 c.f.u.), mutant WbtIG191V (105 c.f.u.), complemented mutant WbtIG191V : : pFNLTP/wbtI (104 c.f.u.) or PBS alone. Fourteen days post-inoculation (PI), the mice were reimmunized with the same doses of the same strains, and 21 days later the mice were challenged IP with 25xLD50 (3x103 c.f.u.), 75xLD50 (9x103 c.f.u.) or 250xLD50 (3x104 c.f.u.) of the LVS parent strain (LD50 120 c.f.u.). For IP immunization, groups of five BALB/c mice each were inoculated with 103, 104 or 5x104 c.f.u. of the mutant in 100 µl of PBS twice 2 weeks apart. Twenty-one days after the second immunization the mice were challenged IP with 75xLD50 (groups immunized with 103 or 104 c.f.u. WbtIG191V) or 250xLD50 (group immunized with 5x104 c.f.u. WbtIG191V) of LVS. Each challenged animal was monitored for 21 days, and severely moribund mice were euthanized. Surviving mice were humanely euthanized using excess CO2, and select tissues cultured for the presence of bacteria, as described above.
Statistical analysis.
The slope of the growth rate was determined from the formula:
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). A C
A transversion in mutant WbtIS187Y caused a codon change of TCTTAT, resulting in the change of Ser to Tyr in residue 187. A GT transversion in mutant WbtIG191V causing a codon change of GGTGTT resulted in residue 191 changing from Gly to Val. The wbtI gene from the LVS was amplified and sequenced, and neither of the substitutions found in the mutants was present in the parent strain. Aligning the amino acid sequence of LVS WbtI with its homologues from 25 species/subspecies indicated that Gly191 was highly conserved in all 25 enzymes (data not shown). Therefore, the phenotype of mutant WbtIG191V and its virulence in mice were further characterized.
Computational structural modelling
The locations of residues Ser187 and Gly191 in WbtI were computationally investigated, as were the effects of S187Y and G191V mutations on enzyme structure (data not shown). Both residues were in the core of the WbtI sugar transamine/perosamine synthetase. The alteration of S187Y or G191V changed the number of -helices, β-sheets and turns of the enzyme, which would result in substantial conformational changes, suggesting that WbtIS187Y and WbtIG191V had lost biological activity.
Physical and chemical characterization of LPS
Since the mutation in WbtIG191V was predicted to be in the O-antigen region, purified LPS samples from WbtIG191V and the parent were separated by electrophoresis and immunoblotted with rabbit polyclonal antiserum to LVS (Fig. 1a) or murine mAb to LPS O antigen (Fig. 1b). A characteristic ladder-like pattern was observed with LVS LPS reacted with both LVS antiserum and mAb, but this pattern was absent from WbtIG191V LPS incubated with either antibody. However, immunoreactive low-molecular-mass material was present in mutant LPS blotted with LVS antiserum, but not with mAb to O antigen, suggesting that the core, but not the O antigen, was present in the mutant. Furthermore, unlike the parental LPS, there was no ladder-like pattern in the LPS of WbtIG191V on polyacrylamide gels stained with Emerald Q fluorescent stain (data not shown). LVS LPS O antigen has been shown to undergo antigenic phase variation to an F. novicida O antigen, resulting in a switch from reactivity to LVS O-antigen antibodies to reactivity with F. novicida O-antigen antibodies (Cowley et al., 1996). However, neither LVS LPS nor WbtIG191V O antigen reacted with antiserum to F. novicida strain U112 in a Western blot, indicating that the mutant did not contain a F. novicida-reactive O antigen. The reactivity of low-molecular-mass LPS from mutant WbtIG191V with F. novicida antiserum indicated conservation of core LPS epitopes between the mutant and F. novicida. The lack of reactivity of low-molecular-mass LVS LPS with F. novicida antiserum was probably due to steric interference of core epitopes by O antigen, further supporting the suggestion that the LPS of WbtIG191V lacked O antigen (Fig. 1c).
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) in WbtIG191V, but no O antigen, was confirmed by MALDI-MS (Fig. 2; details in the legend).
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).
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Serum bactericidal assay
The parental LVS and complemented mutant WbtIG191V : : pFNLTP/wbtI were completely resistant to the bactericidal action of fresh PCS up to at least 40 % (v/v). The LVS (as well as our type A strains Schu S4 and TI0902) and the complemented mutant were also resistant to at least 40 % PCS supplemented with 40 % hyperimmune rabbit serum made to irradiated LVS (data not shown). However, mutant strain WbtIG191V was completely killed by <3 % fresh PCS only, and was much more sensitive to PCS than a non-encapsulated A. pleuropneumoniae control strain. Of interest was that serum susceptibility increased sharply from essentially no killing in 0.5 % PCS to >80 % killing in 1 % PCS (Fig. 4). Similar results were obtained when mouse, canine or human sera were used (data not shown).
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).
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). Therefore, the sensitivity of WbtIG191V incubated with various concentrations of sodium deoxycholate in PBS for 45 min was examined (Fig. 6). On average, in the presence of 0.1 % deoxycholate, more than 100 % of WbtIG191V cells survived (indicating growth of the bacteria during the incubation period), whereas fewer than 60 % of the LVS parental strain cells survived (P=0.02). When the sodium deoxycholate concentration was increased to 1 %, essentially all LVS cells were killed, but 12.4 % of WbtIG191V cells remained viable. Moreover, up to 4.4 % of WbtIG191V mutant cells were able to tolerate up to 10 % deoxycholate. Complemented mutant WbtIG191V : : pFNLTP/wbtI was intermediate between the LVS and the mutant in sensitivity to 0.01 % and 0.1 % deoxycholate, but was as susceptible as the parent at concentrations of deoxycholate of 1 % or more.
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). For this study, mice were inoculated IP with doses of LVS, mutant WbtIG191V or complemented mutant WbtIG191V : : pFNLTP/wbtI that ranged from 200 to 2.8x107 c.f.u. (Table 2). Clinical symptoms were not detected in any of the mice until about 48 h PI, by which time mice inoculated with 2000 c.f.u. LVS had ruffled fur, were inactive and appeared lethargic. All the mice inoculated with this dose of the parental strain died between 4 and 5 days PI. Mice inoculated with lower doses of the parental strain had ruffled fur and were lethargic at 72 h PI. By 6 days PI, all mice inoculated with 600 c.f.u. LVS had died, and three of five mice inoculated with 200 c.f.u. LVS died by 7 days PI. However, by 14 days PI all mice inoculated IP with up to 2.8x105 c.f.u. mutant WbtIG191V remained clinically normal. Mice infected with the two highest doses of the mutant (1.4x106 and 2. 8x107 c.f.u.) showed some early clinical symptoms, such as ruffled fur, stressed breathing and hunched gait, but recovered completely. Mice challenged with 104 c.f.u. complemented mutant WbtIG191V : : pFNLTP/wbtI became moribund within 2 days and died by 4 days PI.
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). Mice challenged with the parental and complemented mutant WbtIG191V : : pFNLTP/wbtI died or were euthanized by the fifth day PI, whereas by 8 days post-challenge all the mice challenged with WbtIG191V appeared normal and had cleared the bacteria from all of their tissues (data not shown).
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). About 3 days post-challenge, the surviving animals developed some clinical symptoms, but later recovered.
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). The surviving mice in this group developed clinical symptoms by day 2 post-challenge, but later recovered. Controls were not immunized IP with LVS because the lethal dose of LVS for mice is low by this route (some mice die from as few as 10 cells). |
DISCUSSION |
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; DeShazer et al., 2001; Harakava & Gabriel, 2003; Liu et al., 2003; Newton et al., 2006; Parsons et al., 2003; Winstanley, 2002), and was therefore used to identify genes uniquely expressed by the more virulent type A and B strains. Seventy-six LVS-specific genes with hypothetical or known functions were identified in eight genomic regions in multiple SSH clones (Ahmed & Inzana, 2004). A putative glycosyl transferase in region VII was chosen for deletion due to its homology to a polysaccharide-biosynthesis gene. This gene was later identified as wbtB, and genomic region VII as the O-antigen locus. Attempts to knock out wbtB using the sacB–CmR cassette in vector pPV resulted in the cointegration of the sacB–CmR gene, as determined by PCR amplification of plasmid DNA from the genome. However, excision of the cointegrant in the presence of sucrose apparently resulted in religation of the original sequence without deletion of wbtB, as PCR and Southern blotting indicated a single, normal copy of wbtB in the genome, but no evidence of plasmid DNA in the genome. Nonetheless, screening of sucrose-resistant strains for lack of iridescence and a dark-red phenotype on Congo red agar (characteristic of F. novicida, but not type A or B strains) resulted in the identification of two LVS mutant strains both containing single-residue (S187Y and G191V) changes in WbtI, as determined by sequencing the O-antigen locus. The G191V mutation in wbtI was confirmed by complementation in trans with a normal copy of the gene, which restored O-antigen synthesis, resistance to the bactericidal action of serum, enhanced susceptibility to sodium deoxycholate, and virulence in mice. Of interest was that complementation occurred in the presence and absence of the groE promoter upstream of wbtI, indicating that a promoter in the plasmid was also likely driving expression of wbtI.
A single amino acid substitution in MglA has been reported to cause the loss of intramacrophage survival and growth of F. novicida (Baron & Nano, 1998). A similar residue alteration appeared to affect the enzymic function of WbtI. Comparative computational modelling showed that Ser197 and Gly191 were both in the core of the wild-type enzyme, and that the side atoms of these residues extended into the milieu. Gly is the smallest and the most flexible amino acid residue. Substitutions or deletions of Gly residues can cause steric hindrance, block the necessary conformational changes in bacterial enzymes, and affect enzyme activity (Li & Rosen, 1998; Li et al., 2000). The wbtI gene is proposed to be a sugar transaminase/perosamine synthetase, required for biosynthesis of 4,6-dideoxy-4-formamido-D-glucose (Prior et al., 2003). Therefore, mutagenesis of this gene should result in complete loss of O antigen, which was confirmed by MALDI-MS analysis. It is not clear why attempts to mutate wbtB resulted in mutations in wbtI. However, the 5' and 3' ends of the O-antigen locus were found to be bordered by the transposase and pseudotransposase IS sequences isftu2 and isftu1, respectively. The presence of these IS sequences may cause the O-antigen locus to be a hypermutable region, resulting in loss of O antigen (the grey colony variant; Hartley et al., 2005), or phase variation to a F. novicida-type O antigen (Cowley et al., 1996).
The loss of polysaccharide capsule or O antigen commonly enhances the susceptibility of Gram-negative bacteria to the bactericidal action of serum (Joiner, 1988). As expected, WbtIG191V was completely killed by fresh 3 % PCS, which lacks antibody, but is rich in complement. In contrast, the parent and complemented mutant were completely resistant to killing by a high concentration of PCS, even in the presence of hyperimmune rabbit serum. Thus, the LPS O antigen is essential to F. tularensis for resistance to the bactericidal activity of serum, and therefore virulence. However, WbtIG191V was still able to grow in the macrophage-like cell line J774A.1, albeit initially at a slower rate than the parental strain. Therefore, the LPS O antigen does not appear to be required for survival inside macrophages, although early intracellular growth was impaired. The susceptibility to normal serum, but resistance to intracellular killing, of WbtIG191V was similar to that of a non-encapsulated mutant of LVS described by Sandström et al. (1988). However, the genetic and biochemical nature of that mutant was not determined. One concern was that mutant WbtIG191V was an LPS phase variant that converted to a F. novicida-like LPS (Cowley et al., 1996). However, the LPS O antigen from this mutant did not react with antiserum to F. novicida or to the parent strain. Another concern was that mutant WbtIG191V was a ‘grey’ colony variant, which has also been reported to lack O antigen (Hartley et al., 2005). There were many phenotypic similarities between the LVS grey-colony variant and WbtIG191V. However, sequence analysis of the wbtI gene of a grey-colony variant that we isolated showed no base change or deletion from that of the parental strain or published LVS genome sequence. Therefore, it appears that any complete loss of O antigen may result in the ‘grey’ phenotype, which as mentioned above, may in part be due to the presence of IS sequences isftu2 and isftu1 upstream and downstream of the O-antigen locus.
Mutant WbtIG191V was more resistant to killing by sodium deoxycholate than the parental strain, which contrasted with results obtained by Cowley et al. (2000), who found that most O-antigen-defective mutants were more susceptible to killing by deoxycholate than the parental strain. However, the mutants tested by Cowley et al. (2000), were derived from F. novicida, which contains an O antigen distinct from that of the LVS. The ability of Gram-negative bacteria to exclude hydrophobic detergents such as deoxycholate depends largely upon the LPS maintaining the stability of the outer membrane through interaction with outer-membrane proteins, and maintaining divalent cations and a hydrophilic cell surface (Cowley et al., 2000). However, the LPS O antigen of type A and B strains is composed entirely of dideoxyglycoses, and contains the sugar 4,6-dideoxy-4-formamido-D-glucose, which is not present in F. novicida (Vinogradov et al., 2004). Furthermore, it was determined that a large amount of the LPS from type A and B strains was extracted into the phenol phase following hot aqueous phenol extraction (unpublished data). Therefore, it is probable that the unusual LPS glycosyl composition of type A and B strains makes the bacterial surface hydrophobic, resulting in enhanced interaction with detergents such as sodium deoxycholate and reduced binding of Congo red.
An important feature of mutant WbtIG191V is that it was highly attenuated in mice following IN challenge, again indicating that the O antigen is required for virulence. The IN route was used to evaluate the capability of each strain to disseminate from the lungs to other tissues, and hence is a better measure of invasiveness than the IP route. Even within 2 days post IN challenge there were more than 2 logs fewer cells of the bacterial mutant present in the tissues than the parental strain, whereas the parental strain and complemented mutant WbtIG191V : : pFNLTP/wbtI continued to multiply in the tissues until the death of the animal. In contrast, the mutant strain continued to diminish in numbers until at sometime after 4 days post-challenge it was completely cleared from all tissues. Since the mutant was not quickly cleared from the tissues, it would be expected that a protective immune response would develop. Although WbtIG191V did induce protection against a relatively low IP challenge dose with the parental strain (25xLD50), less protection was provided against a higher challenge dose (75–250xLD50). However, increasing the immunization dose did increase the resistance to higher challenge doses. The IP route was used for challenge because although not natural, it is the most invasive route and therefore a more sensitive indicator of adaptive immunity, as this route bypasses many aspects of innate immunity. Nonetheless, these results indicate that a complete O antigen does contribute to maximum induction of a protective immune response. Since the LVS strain itself provides only route-dependent protection against challenge with type A F. tularensis (Chen et al., 2003; Conlan et al., 2005; Shen et al., 2004), mice immunized with WbtIG191V were not challenged with a type A strain, but similar studies with a type A mutant are in progress.
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ACKNOWLEDGEMENTS |
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Edited by: N. High
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Baron, G. S. & Nano, F. E. (1998). MglA and MglB are required for the intramacrophage growth of Francisella novicida. Mol Microbiol 29, 247–259.[CrossRef][Medline]
Bates, P. A. & Sternberg, M. J. (1999). Model building by comparison at CASP3: using expert knowledge and computer automation. Proteins (Suppl. 3), 47–54.
Bernier, S. P. & Sokol, P. A. (2005). Use of suppression-subtractive hybridization to identify genes in the Burkholderia cepacia complex that are unique to Burkholderia cenocepacia. J Bacteriol 187, 5278–5291.
Chen, W., Shen, H., Webb, A., Kuolee, R. & Conlan, J. W. (2003). Tularemia in BALB/c and C57BL/6 mice vaccinated with Francisella tularensis LVS and challenged intradermally, or by aerosol with virulent isolates of the pathogen: protection varies depending on pathogen virulence, route of exposure, and host genetic background. Vaccine 21, 3690–3700.[CrossRef][Medline]
Chen, W., Kuolee, R., Shen, H., Busa, M. & Conlan, J. W. (2005). Toll-like receptor 4 (TLR4) plays a relatively minor role in murine defense against primary intradermal infection with Francisella tularensis LVS. Immunol Lett 97, 151–154.[CrossRef][Medline]
Ciucanu, I. & Kerek, F. (1984). A simple and rapid method for the permethylation of carbohydrates. Carbohydr Res 131, 209–217.[CrossRef]
Cole, L. E., Elkins, K. L., Michalek, S. M., Qureshi, N., Eaton, L. J., Rallabhandi, P., Cuesta, N. & Vogel, S. N. (2006). Immunologic consequences of Francisella tularensis live vaccine strain infection: role of the innate immune response in infection and immunity. J Immunol 176, 6888–6899.
Conlan, J. W., Chen, W., Shen, H., Webb, A. & KuoLee, R. (2003). Experimental tularemia in mice challenged by aerosol or intradermally with virulent strains of Francisella tularensis: bacteriologic and histopathologic studies. Microb Pathog 34, 239–248.[CrossRef][Medline]
Conlan, J. W., Shen, H., Kuolee, R., Zhao, X. & Chen, W. (2005). Aerosol-, but not intradermal-immunization with the live vaccine strain of Francisella tularensis protects mice against subsequent aerosol challenge with a highly virulent type A strain of the pathogen by an β T cell- and interferon gamma-dependent mechanism. Vaccine 23, 2477–2485.[CrossRef][Medline]
Cowley, S. C. & Elkins, K. L. (2003). Multiple T cell subsets control Francisella tularensis LVS intracellular growth without stimulation through macrophage interferon 
receptors. J Exp Med 198, 379–389.
Cowley, S. C., Myltseva, S. V. & Nano, F. E. (1996). Phase variation in Francisella tularensis affecting intracellular growth, lipopolysaccharide antigenicity and nitric oxide production. Mol Microbiol 20, 867–874.[CrossRef][Medline]
Cowley, S. C., Gray, C. J. & Nano, F. E. (2000). Isolation and characterization of Francisella novicida mutants defective in lipopolysaccharide biosynthesis. FEMS Microbiol Lett 182, 63–67.[CrossRef][Medline]
Darling, R. G., Catlett, C. L., Huebner, K. D. & Jarrett, D. G. (2002). Threats in bioterrorism. I: CDC category A agents. Emerg Med Clin North Am 20, 273–309.[CrossRef][Medline]
Dennis, D. T., Inglesby, T. V., Henderson, D. A., Bartlett, J. G., Ascher, M. S., Eitzen, E., Fine, A. D., Friedlander, A. M., Hauer, J. & other authors (2001). Tularemia as a biological weapon: medical and public health management. JAMA (J Am Med Assoc) 285, 2763–2773.
DeShazer, D., Waag, D. M., Fritz, D. L. & Woods, D. E. (2001). Identification of a Burkholderia mallei polysaccharide gene cluster by subtractive hybridization and demonstration that the encoded capsule is an essential virulence determinant. Microb Pathog 30, 253–269.[CrossRef][Medline]
Ellis, J., Oyston, P. C., Green, M. & Titball, R. W. (2002). Tularemia. Clin Microbiol Rev 15, 631–646.
Fortier, A. H., Slayter, M. V., Ziemba, R., Meltzer, M. S. & Nacy, C. A. (1991). Live vaccine strain of Francisella tularensis: infection and immunity in mice. Infect Immun 59, 2922–2928.
Golovliov, I., Sjostedt, A., Mokrievich, A. & Pavlov, V. (2003). A method for allelic replacement in Francisella tularensis. FEMS Microbiol Lett 222, 273–280.[Medline]
Hajjar, A. M., Harvey, M. D., Shaffer, S. A., Goodlett, D. R., Sjöstedt, A., Edebro, H., Forsman, M., Byström, M., Pelletier, M. & other authors (2006). Lack of in vitro and in vivo recognition of Francisella tularensis subspecies lipopolysaccharide by Toll-like receptors. Infect Immun 74, 6730–6738.
Harakava, R. & Gabriel, D. W. (2003). Genetic differences between two strains of Xylella fastidiosa revealed by suppression subtractive hybridization. Appl Environ Microbiol 69, 1315–1319.
Hartley, G., Taylor, R., Prior, J., Newstead, S., Hitchen, P. G., Morris, H. R., Dell, A. & Titball, R. W. (2005). Grey variants of the live vaccine strain of Francisella tularensis lack lipopolysaccharide O-antigen, show reduced ability to survive in macrophages and do not induce protective immunity in mice. Vaccine 24, 989–996.[CrossRef][Medline]
Hopla, C. F. & Hopla, A. K. (1994). Tularemia. In Handbook of Zoonoses, pp. 113–126. Edited by G. W. Beran & J. H. Steele. Boca Raton, FL: CRC Press.
Inzana, T. J. (1983). Electrophoretic heterogeneity and interstrain variation of the lipopolysaccharide of Haemophilus influenzae. J Infect Dis 148, 492–499.[Medline]
Inzana, T. J. & Anderson, P. (1985). Serum factor-dependent resistance of Haemophilus influenzae type b to antibody to lipopolysaccharide. J Infect Dis 151, 869–877.[Medline]
Inzana, T. J., Glindemann, G. E., Snider, G., Gardner, S., Crofton, L., Byrne, B. & Harper, J. (2004). Characterization of a wild-type strain of Francisella tularensis isolated from a cat. J Vet Diagn Invest 16, 374–381.
Joiner, K. A. (1988). Complement evasion by bacteria and parasites. Annu Rev Microbiol 42, 201–230.[CrossRef][Medline]
Larsson, P., Oyston, P. C., Chain, P., Chu, M. C., Duffield, M., Fuxelius, H. H., Garcia, E., Hälltorp, G., Johansson, D. & other authors (2005). The complete genome sequence of Francisella tularensis, the causative agent of tularemia. Nat Genet 37, 153–159.[CrossRef][Medline]
Li, J. & Rosen, B. P. (1998). Steric limitations in the interaction of the ATP binding domains of the ArsA ATPase. J Biol Chem 273, 6796–6800.
Li, J. W., Inoue, Y., Miyazaki, M., Pu, H., Kondo, A. & Namba, M. (2000). Growth inhibitory effects of ATP and its derivatives on human fibroblasts immortalized with 60Co-gamma rays. Int J Mol Med 5, 59–62.[Medline]
Li, J., Waters, S. B., Drobna, Z., Devesa, V., Styblo, M. & Thomas, D. J. (2005). Arsenic (+3 oxidation state) methyltransferase and the inorganic arsenic methylation phenotype. Toxicol Appl Pharmacol 204, 164–169.[CrossRef][Medline]
Liu, L., Spilker, T., Coenye, T. & LiPuma, J. J. (2003). Identification by subtractive hybridization of a novel insertion element specific for two widespread Burkholderia cepacia genomovar III strains. J Clin Microbiol 41, 2471–2476.
Maier, T. M., Havig, A., Casey, M., Nano, F. E., Frank, D. W. & Zahrt, T. C. (2004). Construction and characterization of a highly efficient Francisella shuttle plasmid. Appl Environ Microbiol 70, 7511–7519.
Merkle, R. K. & Poppe, I. (1994). Carbohydrate composition analysis of glycoconjugates by gas-liquid chromatography/mass spectrometry. Methods Enzymol 230, 1–15.[CrossRef][Medline]
Newton, H. J., Sansom, F. M., Bennett-Wood, V. & Hartland, E. L. (2006). Identification of Legionella pneumophila-specific genes by genomic subtractive hybridization with Legionella micdadei and identification of lpnE, a gene required for efficient host cell entry. Infect Immun 74, 1683–1691.
Parsons, Y. N., Banasko, R., Detsika, M. G., Duangsonk, K., Rainbow, L., Hart, C. A. & Winstanley, C. (2003). Suppression-subtractive hybridisation reveals variations in gene distribution amongst the Burkholderia cepacia complex, including the presence in some strains of a genomic island containing putative polysaccharide production genes. Arch Microbiol 179, 214–223.[Medline]
Penn, R. L. (2005). Francisella tularensis (tularemia). In Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases, pp. 2674–2685. Edited by G. L. Mandell, J. E. Bennett & R. Dolin. Philadelphia, PA: Elsevier Churchill Livingstone.
Phillips, N. J., Schilling, B., McLendon, M. K., Apicella, M. A. & Gibson, B. W. (2004). Novel modification of lipid A of Francisella tularensis. Infect Immun 72, 5340–5348.
Prior, J. L., Prior, R. G., Hitchen, P. G., Diaper, H., Griffin, K. F., Morris, H. R., Dell, A. & Titball, R. W. (2003). Characterization of the O antigen gene cluster and structural analysis of the O antigen of Francisella tularensis subsp. tularensis. J Med Microbiol 52, 845–851.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sandström, G., Löfgren, S. & Tärnvik, A. (1988). A capsule-deficient mutant of Francisella tularensis LVS exhibits enhanced sensitivity to killing but diminished sensitivity to killing by polymorphonuclear leukocytes. Infect Immun 56, 1194–1202.
Sayle, R. A. & Milner-White, E. J. (1995). RASMOL: biomolecular graphics for all. Trends Biochem Sci 20, 374[CrossRef][Medline]
Shen, H., Chen, W. & Conlan, J. W. (2004). Susceptibility of various mouse strains to systemically- or aerosol-initiated tularemia by virulent type A Francisella tularensis before and after immunization with the attenuated live vaccine strain of the pathogen. Vaccine 22, 2116–2121.[CrossRef][Medline]
Sjöstedt, A. (2005). Family III. Francisellaceae, fam. nov. In Bergey's Manual of Systematic Bacteriology, 2nd edn, pp. 199–210. Edited by G. Garrity, D. J. Brenner, N. R. Kreig, J. T. Staley, D. R. Boone, P. De Vos, M. Goodfellow, F. A. Rainey, G. M. Garrity & K.-H. Schleiffer. New York: Springer.
Staples, J. E., Kubota, K. A., Chalcraft, L. G., Mead, P. S. & Petersen, J. M. (2006). Epidemiologic and molecular analysis of human tularemia, United States, 1964–2004. Emerg Infect Dis 12, 1113–1118.[Medline]
Sturiale, L., Garozzo, D., Silipo, A., Lanzetta, R., Parrilli, M. & Molinaro, A. (2005). New conditions for matrix-assisted laser desorption/ionization mass spectrometry of native bacterial R-type lipopolysaccharides. Rapid Commun Mass Spectrom 19, 1829–1834.[CrossRef][Medline]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Timoney, J. F., Gillespie, J. H., Scott, F. W. & Barlough, J. E. (1988). The genus Francisella. In Hagan and Bruner's Microbiology and Infectious Diseases of Domestic Animals, pp. 132–135. Edited by J. F. Timoney, J. H. Gillespie, F. W. Scott & J. E. Barlough. Ithaca, NY: Cornell University Press.
Titball, R. W. & Sjöstedt, A. (2002). Francisella tularensis: an overview. ASM News 69, 558–563.
Vinogradov, E. V., Shashkov, A. S., Knirel, Y. A., Kochetkov, N. K., Tochtamysheva, N. V., Averin, S. F., Goncharova, O. V. & Khlebnikov, V. S. (1991). Structure of the O-antigen of Francisella tularensis strain 15. Carbohydr Res 214, 289–297.[CrossRef][Medline]
Vinogradov, E. V., Bock, K., Holst, O. & Brade, H. (1995). The structure of the lipid A-core region of the lipopolysaccharides from Vibrio cholerae O1 smooth strain 569B (Inaba) and rough mutant strain 95R (Ogawa). Eur J Biochem 233, 152–158.[Medline]
Vinogradov, E., Perry, M. B. & Conlan, J. W. (2002). Structural analysis of Francisella tularensis lipopolysaccharide. Eur J Biochem 269, 6112–6118.[Medline]
Vinogradov, E., Conlan, W. J., Gunn, J. S. & Perry, M. B. (2004). Characterization of the lipopolysaccharide O-antigen of Francisella novicida (U112). Carbohydr Res 339, 649–654.[CrossRef][Medline]
Ward, C. K., Lawrence, M. L., Veit, H. P. & Inzana, T. J. (1998). Cloning and mutagenesis of a serotype-specific DNA region involved in encapsulation and virulence of Actinobacillus pleuropneumoniae serotype 5a: concomitant expression of serotype 5a and 1 capsular polysaccharides in recombinant A. pleuropneumoniae serotype 1. Infect Immun 66, 3326–3336.
Winstanley, C. (2002). Spot the difference: applications of subtractive hybridisation to the study of bacterial pathogens. J Med Microbiol 51, 459–467.
York, W. S., Darvill, A. G., McNeil, M., Stevenson, T. T. & Albersheim, P. (1985). Isolation and characterization of plant cell walls and cell-wall components. Methods Enzymol 118, 3–40.
Received 26 January 2007;
revised 2 April 2007;
accepted 3 May 2007.
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) LVS parent strain; (*) mutant strain WbtIG191V; (
) complemented mutant WbtIG191V : : pFNLTP/wbtI; (
) serum-susceptible control strain A. pleuropneumoniae J45-100. Each point represents the mean of three experiments. Where error bars are not visible, the SEM was less than 1 %.
) LVS; (
) mutant strain WbtIG191V; (
) complemented mutant strain WbtIG191V : : pFNLTP/wbtI.
