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Bacteriology Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, Frederick, MD 21702, USA
Correspondence
S. L. Welkossusan.
welkos{at}amedd.army.mil
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this paper.
A description of the methods for and results of in silico analysis of the SoaA protein, and a supplementary table listing the oligonucleotide primers used in this study, are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). B. anthracis sporulates in the presence of environmental stresses, such as inadequate nutrient supply or desiccation (Jedrzejas, 2002). The spore, the infectious form of the organism, can remain dormant but viable for decades until favourable conditions are encountered. After infecting an animal host, the spore germinates (Moir et al., 2002), and grows into bacilli that secrete the toxins which ultimately kill the host (Bhatnagar & Batra, 2001; Brossier & Mock, 2001).
The currently licensed human vaccine, anthrax vaccine adsorbed (AVA; Biothrax, Bioport), consists of aluminium hydroxide-adsorbed supernatant material, primarily protective antigen (PA), from cultures of a non-encapsulated, toxigenic strain of B. anthracis. Biothrax is known to be relatively safe (Joellenbeck & Hernandez, 2002) and to protect against both cutaneous and inhalational anthrax (Friedlander et al., 1999; Joellenbeck & Hernandez, 2002; Leppla et al., 2002). Its drawbacks include reactogenicity, the presence of residual lethal factor and oedema factor, which might combine with PA to form active toxins and uncharacterized components, lot-to-lot variation in the amount of PA, and the need for multiple vaccinations to induce and maintain immunity (Brachman et al., 1962). These drawbacks warrant continued efforts to identify novel vaccine strategies. The development of a defined recombinant PA (rPA) vaccine (Farchaus et al., 1998), efforts to reduce the number of doses of AVA required (Pittman et al., 2002), and development of better potency assays (Little et al., 2004) have alleviated some of the problems associated with the current human vaccine; however, there are compelling reasons to warrant development of a new-generation vaccine.
Despite the availability of PA-based vaccines and effective antibiotic therapy against B. anthracis (Friedlander et al., 2002), the potential exists for the emergence of naturally existing or genetically engineered strains which are antibiotic-resistant and/or refractory to PA vaccine-induced immunity. To counteract the threat of such potential bioweapons, effective alternative vaccines and therapies should be developed. Better protection might be achieved, for instance, by targeting the early stages of infection by the spore as well as the toxins produced by the vegetative bacilli (Brossier et al., 2002; Cohen et al., 2000; Cote et al., 2005; Welkos et al., 2002). Thus, we hypothesized that a combination of PA and a defined set of spore-associated proteins could contribute to the development of a less reactogenic and more efficacious anthrax vaccine to provide broader coverage against emerging and/or engineered threats.
By characterizing novel spore antigens or defined antigens of unknown immunological significance, we hope to better understand not only spore biology but also the host response to such antigens. In this report, we characterized a protein that is unique to the Bacillus cereus group. We found that this protein was associated with spore opsonization and refer to it as spore opsonization-associated antigen A (SoaA).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Escherichia coli strains DH5
and GM2163 were used for cloning and mutant construction (Table 1). Libraries of B. anthracis Ames strain mini Tn10 insertion mutants were constructed by transposition mutagenesis, as described previously (Day et al., 2007; Gominet et al., 2001; Steinmetz & Richter, 1994). When appropriate, antibiotics were used at the following concentrations: kanamycin (50 µg ml–1 for E. coli and 20 µg ml–1 for B. anthracis), spectinomycin (125 µg ml–1 for E. coli and 250 µg ml–1 for B. anthracis), and erythromycin (0.4 or 5 µg ml–1 as indicated for B. anthracis). Restriction enzymes and T4 DNA ligase were purchased from New England BioLabs and were used in accordance with the manufacturer's instructions. Pathogen-free BALB/c mice were obtained from the National Cancer Institute, Fort Detrick (Frederick, MD). Pathogen-free Hartley guinea pigs were obtained from Charles River Laboratories.
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, 2005; Welkos et al., 1989). Where indicated, spores were germinated by exposure to a medium containing L-alanine, adenosine and casamino acids (AAC) medium (Welkos et al., 2004) for 1–2 h at 37 °C. Anti-spore antibodies (i.e. antibody preparations 733 and 1250) were obtained from the sera of New Zealand white rabbits (Covance) vaccinated with purified preparations of ungerminated spores of the Ames strain that had been inactivated by gamma irradiation before injection, as described previously (Welkos et al., 2004). In addition, a mAb (BA-MAB5) was used for immuno-electron microscopy, Western blots and phagocytosis assays. This mAb was purchased from the Department of Defense Critical Reagent Program maintained under the Joint Program Executive Office for Chemical and Biological Defense. This antibody was provided to the Critical Reagent Program by the Identification Group, Detection Department, Defence Science and Technology Laboratory (Dstl), Porton Down, Wiltshire, UK. This antibody, BA-MAB5, was developed against unfixed purified exosporia (Redmond et al., 2004) from the B. anthracis Ames strain. While the precise epitope of BA-MAB5 has not been mapped, it recognizes a protein of 
250 kDa and is presumed to be BclA. Further characterization of the target of BA-MAB5 in this study demonstrates that this mAb does target BclA (Results, Fig. 2 g, h and Western blots; data not shown).
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; Nicholson & Setlow, 1990). In the modified procedure, the vegetative library was sporulated by culture in Leighton–Doi broth with spectinomycin, and the spores were harvested and purified as described above. Single colonies of the spore library were inoculated by patching onto a filter placed on a new sporulation medium (NSM) agar plate (Phillips & Ezzell, 1989) containing spectinomycin. After patching 50 colonies to each plate, the latter were wrapped in foil and incubated at 37 °C. After 24 h at 37 °C, they were transferred to room temperature for several days to complete sporulation, which was determined by phase-contrast microscopy. The filters were each then transferred to agar plates containing D-alanine (Nicholson & Setlow, 1990; Clements & Moir, 1998) and the plates were heated in an oven for 2 h at 65 °C. The filters were then transferred to plates of germination agar (GA), prepared as described elsewhere (Clements & Moir, 1998) or after supplementation with AAC medium (Welkos et al., 2004). The filters on the GA were overlaid with molten 0.8 % soft agar containing anti-spore antibodies and the plates were then incubated at 37 °C. The patch plates were screened for mutants that germinated more rapidly than the wild-type parental strain in the presence of the antibodies and thus formed red colonies in contrast to the white or pink colonies formed by isolates that were more sensitive to antibody-mediated germination inhibition or were slower to germinate. In this method, germinating spores were detected by their ability to reduce the colourless tetrazolium dye contained in the GA medium to a red formazan derivative (Nicholson & Setlow, 1990; Clements & Moir, 1998).
Characterization of the germination of library mutants.
The rate and extent of germination of the library mutants compared to those of the parent strain were assessed further in several assays, including the microtitre spectrofluorometric assay of fluorescent dye uptake during germination (Welkos et al., 2004), determination of heat resistance (Welkos et al., 2004), determination of changes in refractility by phase-contrast microscopy (Welkos et al., 2004), and measurement of changes in OD590 (Clements & Moir, 1998).
Cloning and identification of mutated sequences.
The transposon-disrupted gene sequences in selected mutants were identified by standard rescue cloning procedures. Chromosomal DNA was isolated from the mutant B. anthracis strain as described elsewhere (Keim et al., 1997; Jackson et al., 1997) and digested with HindIII, and the digested DNA was self-ligated. The ligated DNA was butanol-precipitated and electroporated into E. coli. Plasmids were isolated from selected colonies on spectinomycin-containing agar plates and the plasmids were sequenced using the Thermo Sequenase Dye Terminator Cycle Sequencing pre-mix kit (Amersham BioScience). The sequences obtained were compared to those in the GenBank database. As determined by BLAST searches of the cloned DNA versus the GenBank entry (accession no. 47530577) for the B. anthracis strain Ames genome, the transposon insertion described in this paper occurred in an ORF that encodes a protein of unidentified function (B. anthracis hypothetical protein BA5269).
Construction of mutant B. anthracis strains
B. anthracis soaA : : Kan.
A 1.5 kb DNA fragment containing the soaA gene was PCR-amplified using the primer pair SoaA-NotI and SoaA-AscI (see Supplementary Table S1 available with the online version of this paper) from Ames chromosomal DNA, and cloned into the shuttle vector pEO-3, as detailed previously (Bozue et al., 2005; Mendelson et al., 2004), and the soaA gene was interrupted with the 
-Kan-2 fragment (Perez-Casal et al., 1991) at a unique PmlI site within the gene. The Ames strain of B. anthracis was then transformed with the plasmid containing soaA : : Kan as described previously (Mendelson et al., 2004). The plasmid was next integrated into the chromosome and the cointegrates were selected on the basis of antibiotic (kanamycin) and temperature (42 °C) resistance (Mendelson et al., 2004). Eight kanamycin-resistant colonies were selected and streaked onto two separate plates each containing an antibiotic: kanamycin (to select for the presence of the -Kan-2 fragment) and erythromycin (5 µg ml–1; to detect pEO-3). One of the clones was resistant to kanamycin but sensitive to erythromycin. This putative mutant clone, strain Ames soaA : : Kan, was confirmed by PCR analysis using the primer pairs SoaA 5' and OL289 and SoaA3' and OL289 listed in Supplementary Table S1.
Construction of the cylI : : Kan B. anthracis mutant.
A 3 kb DNA fragment containing the cylI gene (BA5268), which encodes a putative cytoloysin immunity domain protein with homology to the CylI protein from Enterococcus faecalis (Coburn et al., 1999), was PCR-amplified from Ames strain chromosomal DNA and cloned into vector pEO-3 using the primer pair CylI-NotI and CylI-AscI. The cloned cylI gene was inactivated at the MfeI restriction enzyme site by inserting the -Kan-2 fragment. The plasmid was transformed and integrated into the chromosome of the Ames strain as described above. Putative cylI mutants in the Ames strain were confirmed by PCR analysis using the primer pairs CylI5' and OL289 and CylI 3' and OL289 listed in Supplementary Table S1.
RT-PCR analyses.
RNA was isolated and RT-PCR was performed using the One-step RT-PCR kit (Qiagen) as described previously (Cote et al., 2005). The primer pair SoaA-Lower and SoaA-Upper was used to detect mRNA from the soaA gene. The primer pair Cyto-Lower and Cyto-Upper was used to detect mRNA from the cylI gene. The primer pair SoaA-intergenic and Cyto-intergenic was used to verify that the soaA and cylI genes were located on a single transcript. The primer pair SoaA5' and BA5270 was used to show that the BA5270 gene and the soaA gene were located on a single transcript. Primers used in the RT reactions are included in Supplementary Table S1.
Macrophage assays for spore phagocytosis and intracellular viability.
RAW264.7 cells were cultured in 24-well trays with coverslips and used in phagocytosis and intracellular spore viability assays as described previously (Cote et al., 2005; Welkos et al., 2001, 2002). The cell cultures were infected with spores at an m.o.i. of 1–2 spores per macrophage. To promote spore binding and phagocytosis by the macrophages, the infected cell cultures were centrifuged at low speed at 260 g for 30 min at 30 °C and incubated for an additional 30 min at 37 °C in 5 % CO2. As described by Banks et al. (2005), they were then incubated for 30 min at 37 °C in medium containing 10 % fetal bovine serum and 5 µg gentamicin ml–1 then extensively washed to remove most of the unphagocytosed spores. The effects of antibodies on phagocytosis and intracellular spore germination and viability were assayed bacteriologically by viable-count determinations and by light and immunofluorescence microscopy of stained samples (Cote et al., 2005; Welkos et al., 2001, 2002). Samples were stained with spore stain (malachite green) and counterstained with a Wright–Giemsa stain (Diff-Quik) (Welkos et al., 1989) for light microscopy. For immunofluorescence microscopy, coverslips were incubated with a rabbit antibody prepared against whole killed spores of the Ames strain that recognizes ungerminated and germinated spores as well as bacilli, and the spores were detected by double-labelling with secondary antibodies, as described previously (Cote et al., 2005; Welkos et al., 2002).
Protein purification and antibody production.
Recombinant proteins were produced by the James Madison University Center for Integrated Science and Technology (Harrisonburg, VA). Several strategies were employed to express and purify the SoaA protein. The ORF encoding the full-length SoaA protein (236 aa) was amplified and cloned into the protein expression vector pTYB2 (New England Biolabs) using primers GBAAf1 and GBAAr236 (Supplementary Table S1). Because the untagged full-length protein was insoluble (data not shown), several different fragments of the SoaA protein were cloned into pTYB12 (IMPACT-CN system, New England Biolabs) to express a soluble portion of the protein by creating a protein–intein fusion product. The fusion proteins were purified over a chitin column, which bound the intein tag (Goodin et al., 2005). DNA fragments encoding the full-length protein (amino acids 1–236), or amino acids 35–236, 141–193 or 141–236, were amplified and cloned into pTYB12. The 6 kDa peptide consisting of amino acids 141–193 was soluble; however, the other three fusion proteins remained insoluble. The DNA encoding the soluble peptide (referred to as 6 kDa SoaA) was amplified by using the primers GBAAf141 and GBAAr193 (Supplementary Table S1). Full-length (untagged) SoaA protein was purified as inclusion bodies using B-PER bacterial protein purification reagent as described by the manufacturer (Pierce Biotechnology) and the 6 kDa SoaA peptide was expressed and purified as an intein fusion protein (Goodin et al., 2005). Based on hydropathy and antigenicity plots, this segment was predicted to be highly antigenic.
Rabbits were vaccinated (by Covance as described above) with either the insoluble inclusion bodies containing the SoaA protein or the soluble 6 kDa SoaA peptide. The Ribi-adjuvant system (Corixa) was used and the vaccination strategy was that recommended by the manufacturer. The antisera obtained were IgG-purified, as described previously (Welkos et al., 2004). In addition, prebleed rabbit sera (collected before vaccination) were also IgG-purified and served as negative controls in our assays, where indicated.
Electron microscopy.
Immuno-electron microscopy was used to observe spores as described previously (Cote et al., 2005). The sections were incubated in primary antibody after it had been normalized to 1 mg ml–1 and diluted 1 : 100 (final concentration 10 µg ml–1). The primary antibodies included rabbit anti-spore polyclonal antibody 733, rabbit anti-SoaA polyclonal antibody, rabbit anti-6 kDa/SoaA antibody and normal pre-immune rabbit IgG as a negative control. Negative controls were normal non-immune rabbit IgG. Additional sections not treated with primary antibody were prepared to test the specificity of secondary antibodies. All sections were treated with blocking buffer and gold-labelled secondary antibodies.
Construction of GFP fusions.
A GFP fusion vector pEO-3-gfp was constructed by cloning a KpnI/HindIII fragment containing the gfp-mut2 gene (Lemon & Grossman, 1998) into the KpnI/HindIII site of pEO-3 (Bozue et al., 2005; Mendelson et al., 2004). To construct a translational C-terminal fusion of SoaA to GFP, the soaA gene was amplified using primers soaA-GFP 3' and soaA-KpnI GFP 5' (Supplementary Table S1) utilizing Phusion DNA polymerase (New England Biolabs). The PCR product was cloned into the KpnI/PmeI sites of vector pEO-3-gfp. The resultant plasmids were passaged through E. coli strain GM2163 and electroporated into the Ames strain of B. anthracis. The plasmid was cointegrated into the chromosome to produce a translational fusion.
Protein electrophoresis and Western blotting.
Spore extracts were prepared by homogenizing spores in extraction buffer containing urea [0.05 M dithioerythritol, 1 % SDS, 8 M urea in 0.005 M cyclohexyl aminomethane sulfonic acid (CHES), pH 9.8; UDS] (Pandey & Aronson, 1979). Briefly, spores were suspended in UDS and homogenized in a Bio 101 Fast Prep (Qbiogene) using lysing matrix B tubes. The spore lysate was transferred to a fresh tube and centrifuged, and the supernatant fluid was saved for further analysis. The proteins in the supernatant fluids were separated on a 4–20 % Tris-glycine mini gel (Invitrogen) and stained with Gel Code Blue (Pierce). The proteins were electroblotted onto PVDF membranes in 20 % methanol, 0.3 % SDS and Tris-glycine transfer buffer (Pierce). Membranes were blocked in a 5 % non-fat milk solution and then incubated with a commercially available affinity-purified rabbit anti-GFP polyclonal serum (ICL) at a dilution of 1 : 2000, or polyclonal IgG directed against gamma-irradiated spores at a dilution of 1 : 500. After washing, the membranes were incubated with an anti-rabbit horseradish peroxidase-conjugated secondary antibody diluted 1 : 4000 or 1 : 10 000 (Pierce). Immunoreactive bands were visualized using 4-chloronaphthol/3,3'-diaminodbenzidine (4CN/DAB) substrate (Pierce).
Comparisons of in vitro resistance and growth.
Spores were subjected to several adverse conditions to ascertain the resistance properties of the soaA : : Kan mutant spores compared to those of the Ames parent spores. Spores were exposed to 100 % methanol, proteinase K (30 µg ml–1), lysozyme (250 µg ml–1), HCl (1, 0.5 and 0.25 M), NaOH (1, 0.5 and 0.25 M) or chloroform (10 %) for 30 min. The spores were then washed and plated to assess viability. In addition, the effect of extended periods at elevated temperatures was also examined. For this assay, spores were incubated at 70 °C and aliquots were removed to determine the amount of time required to achieve a 90 % loss of viability (Bozue et al., 2005).
To compare the in vitro growth rates of the wild-type Ames and soaA : : Kan strains, 25 ml LB cultures were each inoculated with 5x106 spores of both strains. The cultures were incubated at 37 °C with aeration and aliquots were removed at selected time points (0, 2, 6 and 10 h). The aliquots were mixed by vortexing, diluted then plated onto LB plates with or without kanamycin.
Effects of the soaA : : Kan mutation on virulence and in vivo fitness.
To assess potential alterations in virulence associated with the soaA : : Kan mutation, we utilized both a mouse intraperitoneal model and a guinea pig intramuscular model of infection. Mice were challenged intraperitoneally with varying doses of spores (Cote et al., 2004). Guinea pigs were challenged intramuscularly (Bozue et al., 2005; Fellows et al., 2001) with approximately five LD50 doses (Ames equivalents) of either wild-type Ames strain spores or soaA : : Kan spores. The guinea pig in vivo fitness model was utilized as described previously (Bozue et al., 2007). Briefly, Hartley guinea pigs were challenged intramuscularly with approximately equal challenge doses of wild-type Ames spores and mutant spores (either soaA : : Kan or cylI : : Kan). Two days later, spleens were harvested from moribund guinea pigs and the bacterial load was quantified for each spleen. The relative percentage recovery was calculated by determining the percentage of mutant bacteria (based upon antibiotic resistance) recovered from the spleen relative to the percentage of mutant spores present in the initial challenge dose. The same procedure was also performed with infections initiated with a single B. anthracis strain (either wild-type Ames or soaA : : Kan).
Passive protection studies.
Approximately 12-week-old female BALB/c mice received intraperitoneal injections of purified rabbit IgG that was directed against whole irradiated spores (1250) or against recombinant SoaA (anti-SoaA), or normal pre-immune IgG. Another group received PBS as a control. The IgG (300 µg) was injected approximately 5 h prior to an intraperitoneal spore challenge with approximately five lethal doses of B. anthracis Ames strain spores (Popov et al., 2004). Mice were observed for 14 days.
Statistical and sequence analysis program tools.
Survival rates were compared between each treatment group and the control group by Fisher exact tests with permutation adjustment for multiple comparisons. Kaplan–Meier/product-limit estimation was used to construct survival curves and to compute mean survival times. Survival curves were compared between each treatment group and the control group by log rank tests with Hochberg adjustment for multiple comparisons. Mean times-to-death were compared between each treatment group and the control group by t tests with permutation adjustment for multiple comparisons. The above analyses were conducted using SAS version 8.2 (SAS OnlineDoc version 8; SAS Institute). The in vitro germination kinetics of spores in different peritoneal fluids was analysed by a four-parameter logistic regression model available in the SigmaPlot PC software program (Welkos et al., 2004). When comparing relative bacterial counts recovered from spleens, statistical significance (P<0.05) was determined by the two-tailed Student's t test with the GraphPad Prism software (GraphPad). DNA sequence analysis was performed using DNASTAR and the NCBI BLASTP program was used to identify homologues in other bacteria.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Welkos et al., 2004). The soaA gene (BA5269), encoding a ‘hypothetical protein’, was 724 bp in length and was 43 bp upstream of a gene (BA5268) that shares homology to the cytolysin immunity gene (cylI) from Ent. faecalis. In Ent. faecalis, the CylI protein protects the bacterium from being damaged by the cytolysins that it produces (Coburn et al., 1999) (Fig. 1a). The CylI homologue in B. anthracis exhibited approximately 29 % identity and 47 % similarity with the Ent. faecalis CylI protein, as well as significant identity with homologues in B. cereus and Bacillus thuringiensis (99 and 94 %, respectively). In addition, a small ORF (BA5270) was located immediately upstream of the soaA gene. This gene was shown to have significant homology with putative DNA-binding proteins from B. cereus (97.5 % identity) (Rasko et al., 2004) and B. thuringiensis (98.7 % identity) (Challacombe et al., 2007). These three genes shown in Fig. 1(a) (BA5270, 5269 and 5268) are maintained in the same order in B. cereus (BCE-5169, 5168 and 5167) and B. thuringiensis (BT 9727-4739, 4738 and 4737). BA5270 is in a different reading frame to soaA, but the coding sequences of these genes overlap by 15 nt (Johnson & Chisholm, 2004). A putative sigma-K (
K)-dependent promoter sequence (Amaya et al., 2001) was located upstream of BA5270 (Fig. 1b). RT-PCR demonstrated that the BA5270, soaA and cylI genes could be cotranscribed (data not shown).
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Localization of the SoaA protein
Electrophoretic analyses comparing whole-spore extracts from wild-type Ames spores and soaA : : Kan spores did not reveal any changes in spore coat protein profiles (data not shown). Using polyclonal antibodies directed against recombinant SoaA (and the 6 kDa hydrophilic portion of SoaA), the protein was localized to the area of the cortex underneath the spore coat of the ungerminated wild-type spore by immuno-electron microscopy (Fig. 2a
). Ungerminated spores contained more of the SoaA protein than germinated spores, as suggested by the decrease in staining of the latter (112±25 gold particles per spore, n=18 spores, Fig. 2a, and 33±12 gold particles per spore, n=17 spores, Fig. 2b, respectively). These differences in the presence of SoaA on the spores were statistically significant (P<0.001). These data suggest that the SoaA protein is either degraded or released to the surface during germination. The soaA : : Kan mutant spores retained negligible levels of binding to anti-SoaA antibodies (15±7 gold particles per spore, n=18, Fig. 2c). This low level of binding could be due to the production of a predicted small 72 aa peptide in our mutant strain. It is possible that this peptide was produced and detected by the polyclonal anti-SoaA antibodies. This small peptide, however, did not contain the proposed immunodominant 6 kDa peptide of the SoaA protein.
Further immuno-electron microscopy analyses illustrated that the anti-SoaA staining pattern is different from that observed using an anti-spore IgG. This anti-spore IgG is directed predominantly against the BclA protein (the immunodominant protein of B. anthracis spores described by Steichen et al., 2003; Sylvestre et al., 2002), which is located on the surface of the spore and forms the ‘hair-like’ projections from the exosporium (Fig. 2d). The soaA : : Kan mutant spores also stained similarly with the anti-spore antibodies (Fig. 2e). Lastly, we examined spores of the BclA knockout mutant of the Ames strain (bclA : : Kan) (Bozue et al., 2007) with anti-whole spore IgG (Fig. 2f). These data obtained from micrographs confirmed that the BclA protein is immunodominant in the immune response generated against gamma-irradiated spores, but also reaffirmed that other non-BclA antigens are recognized (Fig. 2f).
One possible effect of the absence of a functional SoaA protein could be alterations in the expression of BclA on the surface of the spores. To further characterize potential alterations in the expression of BclA in the soaA : : Kan mutant spores, wild-type Ames spores and soaA : : Kan spores were stained with a mAb against BclA (Fig. 2g, h, respectively). It was determined that there was no significant difference in binding of the mAb to wild-type spores or soaA : : Kan spores (79±24 gold particles, n=22, and 74±22 gold particles, n=16 for wild-type and soaA : : Kan spores, respectively). It is also noteworthy that all of the staining patterns observed in these electron micrographs, described above, were significantly different from that for pre-bleed IgG (2.3±2.8 gold particles per spore, n=30 spores, Fig. 2i).
Western blot analyses were also carried out to identify any putative differences in BclA expression that could be attributed to the soaA : : Kan genotype. When either polyclonal or monoclonal antibodies were used, the Western blots indicated no appreciable differences in BclA expression on the soaA : : Kan spores compared to the wild-type spores, regardless of whether the spores were refractile or germinated (data not shown).
To further characterize the location of the SoaA protein, we created a B. anthracis strain containing a translational SoaA : : GFP fusion. Experiments involving the SoaA : : GFP fusion illustrated that soaA expression was mainly associated with sporulating cultures. Non-sporulating exponential-phase vegetative cultures did not express GFP (Fig. 3a, two left panels). However, cultures that had begun to sporulate (overnight stationary-phase cultures) did exhibit GFP expression (Fig. 3a, two right panels, and Fig. 3b). GFP was most obviously expressed in bacilli without a clearly visible mature spore, suggesting that the SoaA protein is incorporated into the forespore by the mother cell relatively early in spore assembly (Fig. 3b). This was also predicted by the presence of a putative K-dependent promoter sequence (Fig. 1a) which is associated with gene expression during sporulation (Helmann & Moran, 2002).
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55 kDa) in the Ames spores containing the SoaA : : GFP fusion, but not in the wild-type spores (Fig. 3c). Western blot analyses also detected a band representing GFP alone. This GFP band is likely to be the result of sporulation-associated proteases that cleave the GFP domain from the SoaA : : GFP fusion construct. Similar instances of the cleavage of the GFP domain from GFP fusion constructs have been reported previously (Kim et al., 2006; Little & Driks, 2001). SoaA : : GFP concentrations in extracts prepared from ungerminated spores were below the level of detection by Western analysis (data not shown). However, IgG directed against gamma-irradiated ungerminated spores did react with the 6 kDa recombinant peptide that was shown to be hydrophilic and likely antigenic (Fig. 3d). It has been shown that gamma irradiation induces significant membrane permeabilization in Bacillus spores (Laflamme et al., 2004). Thus, we hypothesize that the SoaA protein present beneath the coat in ungerminated spores becomes available to the immune system upon gamma irradiation. Conceivably, this IgG directed against gamma-irradiated spores may better represent the entire pool of spore epitopes that could be seen by the infected host from spore inhalation to spore germination and eventual outgrowth.
Effects of the soaA : : Kan mutation on spore resistance properties
Spores were exposed to HCl, NaOH, lysozyme, proteinase K, methanol, chloroform and extended periods at an elevated temperature. There were no significant differences observed in the resistance properties of the soaA : : Kan spores as compared to Ames parental spores under any conditions tested (data not shown). These results suggest that the SoaA protein does not contribute significantly to the resistance of spores exposed to these deleterious experimental conditions.
Effects of the soaA : : Kan mutation on spore germination
As determined by phase-contrast microscopy (Welkos et al., 2004), spectrofluorometric germination assays (Welkos et al., 2004) and the decrease in OD (Paidhungat & Setlow, 1999), there were no significant differences in in vitro germination rates of the Ames wild-type and soaA : : Kan spores (data not shown). However, the wild-type and mutant spores differed in their germination potential when loss of heat resistance was used as a germination marker. Briefly, spores were exposed to germinants for selected amounts of time, and then exposed to 65 °C for 30 min. The heated samples were then diluted and the dilutions plated to assess spore viability after heating (Welkos et al., 2004). The ability of the soaA : : Kan mutant to germinate more readily than the wild-type as reflected by the loss of heat resistance (loss of viability) was observed to be statistically significant in five individual experiments. In one representative experiment, after a 5 min exposure to the germination medium, the wild-type Ames spores were determined to be 73.6 %±2.3 % germinated (heat-sensitive), while the soaA : : Kan spores were determined to be 89.2 %±1.4 % germinated (heat-sensitive) (P=<0.0001). In contrast, as detected in these germination assays, the cylI : : Kan mutant germinated to the same extent as the wild-type Ames strain (data not shown). These results suggested that a secondary reduction in expression of CylI was probably not the cause of phenotypic changes associated with disruption of the soaA mutant.
Effects of the soaA : : Kan mutation on spore opsonization
Antibodies directed against gamma-irradiated ungerminated spores (antibody 733) can have opsonic effects on wild-type Ames spores, resulting in an enhancement of phagocytosis compared to that of wild-type spores treated with prebleed IgG (Fig. 4a; S. L. Welkos and others, unpublished data). In contrast, as observed with the transposon mutant originally isolated, the antibody exhibited little ability to enhance phagocytosis of the soaA : : Kan mutant spores (Fig. 4a). These conclusions were supported by viable count determinations of infected macrophages (Fig. 4) and also microscopically, by spore and histological staining and by double-label fluorescent immunostaining (data not shown).
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). In addition, using the IgG fraction from antisera directed against a small hydrophilic portion of the SoaA protein (anti-6 kDa-SoaA), we observed the same enhanced opsonization associated with these antibodies as we observed with antibodies to whole spores and to the full-length SoaA protein (Fig. 4c). We also examined the opsonization of bclA : : Kan spores by the anti-spore antibody (733). Spores of the bclA : : Kan mutant were also not significantly opsonized by the anti-spore antibodies (Fig. 4d).
We also determined the effect of anti-SoaA antibodies on the phagocytosis of bclA : : Kan spores. As predicted by earlier experiments, the bclA : : Kan spores are not opsonized by anti-SoaA antibodies, although wild-type spores are (Fig. 4e). These results were expected, as IgG directed against whole irradiated spores did not opsonize the bclA : : Kan spores (Fig. 4d). To further explore the possible relationship between SoaA and BclA, we examined the effects of an anti-BclA mAb (MAB-5) on the phagocytosis of soaA : : Kan spores (Fig. 4f). This mAb was opsonic for both wild-type and soaA : : Kan spores. Microscopy and protein analysis suggested that soaA : : Kan spores retain native levels of BclA (Fig. 2 g, h; data not shown).
In addition, we examined the effects of anti-PA antibodies on the opsonization of the soaA : : Kan spores. Pretreating soaA : : Kan spores with anti-PA IgG significantly increased the phagocytic uptake of the soaA : : Kan spores (data not shown). This anti-PA antibody-associated enhanced uptake of the mutant spores was similar to that observed with wild-type Ames spores, as shown previously (Cote et al., 2005; Welkos et al., 2002). This result suggests that the differences between the soaA : : Kan spores and wild-type spores in their responses to the opsonizing activity of anti-spore antibodies are likely to be specific to IgG directed against the exosporium and/or other spore components and not to IgG directed against PA.
Effects of the soaA : : Kan mutation on virulence and in vivo fitness of the bacterium
To assess potential alterations in virulence associated with the soaA : : Kan mutation, we utilized both a mouse intraperitoneal (Cote et al., 2004; Popov et al., 2004) and a guinea pig intramuscular model of infection (Bozue et al., 2005; Fellows et al., 2001). All guinea pigs challenged with approximately five Ames LD50 equivalents of either wild-type Ames or soaA : : Kan spores succumbed to the ensuing infection within 4 days (data not shown). In our mouse intraperitoneal model of infection, varying doses were administered (ranging from approximately 0.5 Ames LD50 equivalents to six Ames LD50 equivalents). There was no statistically significant difference noted in virulence between the wild-type spores and the soaA : : Kan spores (data not shown).
To further explore the potential phenotypes associated with spores of the mutant soaA : : Kan strain, we employed an in vivo competition assay. Guinea pigs were infected with approximately equal challenge doses of Ames parent spores and mutant spores (soaA : : Kan or cylI : : Kan). Two days post-challenge, moribund guinea pigs were euthanized and the spleens were harvested. Bacterial loads in the spleens were determined, and the ratio of parent to mutant bacilli recovered (based on antibiotic resistance) was determined.
The cylI : : Kan strain was able to survive in vivo at comparable levels to those observed for the Ames parental strain (Fig. 5). Interestingly, however, the soaA : : Kan strain appeared to be significantly less suited for survival in vivo (P<0.0001) compared to the wild-type Ames strain (Fig. 5). When control competitive experiments were performed in vitro with the Ames strain and the soaA : : Kan mutant strain, no differences in bacterial growth and survival were observed (data not shown).
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Passive protection using rabbit IgG directed against spore epitopes
Passive protection studies were carried out to determine if rabbit IgG directed against irradiated spores or SoaA would offer protection from an intraperitoneal infection with B. anthracis spores. As shown in Fig. 6, there was significant protection afforded to the mice by anti-irradiated spore IgG when compared to a PBS buffer control (percentage survival P=0.0296 and survival curve P=0.0153). However, when compared to IgG purified from pre-immune normal rabbit sera, these differences were modest and statistically insignificant (P>0.05). Anti-SoaA IgG did not significantly protect the mice when compared to either the PBS or the pre-immune IgG controls (P>0.05).
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DISCUSSION |
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). The mutation in this gene was isolated during our attempts to identify proteins that were recognized in vitro by antisera directed against spore entities with potential roles in the early events of in vivo spore germination and the host response.
The soaA mutant was isolated from a library of transposon-mutagenized Ames spores and was identified in a screen for mutants resistant to the germination inhibition activity of anti-spore antibodies. To analyse its characteristics in detail, an allelic exchange mutation was made in the soaA gene. The soaA : : Kan mutant strain of B. anthracis Ames strain exhibited several interesting phenotypes. The SoaA protein appeared to be deposited onto the spore early in sporulation (Fig. 3), and in ungerminated spores the SoaA protein was located in the area of the cortex underneath the spore coat, possibly the inner spore membrane (Fig. 2a). Our data suggest that the SoaA protein was released, degraded during germination (Fig. 2b), or exposed by some other mechanism on or near the surface of the germinating spore in a manner that affected spore–macrophage interactions. This release or degradation of SoaA appeared to coincide with the hydrolysis of the spore cortex during germination.
It has been noted that the inner membrane of the spore expands significantly during germination. The inner spore membrane of Bacillus species can expand as much as twofold during the first minutes of germination (Cowan et al., 2004). The SpoVAD protein described by Vepachedu & Setlow (2005) is an integral membrane protein with seven transmembrane domains that is localized to the inner spore membrane of Bacillus subtilis. Although the SpoVAD protein shares no homology with the SoaA protein, the authors noted that there was an approximately two- to threefold decrease in the amount of SpoVAD found in germinated/outgrown spores as compared to dormant spores (Vepachedu & Setlow, 2005). This observation is similar to our data regarding SoaA levels in germinated spores compared to ungerminated spores of B. anthracis.
However, Western blot analyses detected the SoaA : : GFP fusion in germinated spores (Fig. 3d) but not in ungerminated spores (data not shown). We believe that our extraction procedures are adequate to lyse the ungerminated spores, as we can efficiently detect nucleic acids from these lysed spore samples (Cote et al., 2005). One hypothesis concerning this apparent failure of SoaA detection via Western blot analyses of ungerminated spores is that the SoaA protein is cross-linked to other spore components and in a form that cannot be readily resolved by SDS-PAGE until germination is initiated.
We hypothesized that this protein plays some role in antigen expression on the surface of the spore upon initiation of germination, thus affecting spore and anti-spore IgG interactions. Importantly, however, the soluble 6 kDa SoaA peptide was recognized by IgG directed against gamma-irradiated ungerminated Ames spores (Fig. 3d), confirming that the SoaA protein is associated with ungerminated spores and was possibly made accessible to the immune system upon gamma irradiation (Laflamme et al., 2004).
We observed that spores of the soaA : : Kan strain were not readily opsonized in the presence of rabbit polyclonal anti-spore IgG, whereas the phagocytosis of wild-type spores was significantly stimulated in the presence of such antibodies (Fig. 5a). The antibodies directed against ungerminated spores were composed predominantly of antibodies to the immunodominant exosporium antigen BclA (Fig. 2c, d), although other spore antigens were also recognized (Fig. 2f) (Steichen et al., 2003, 2005; Sylvestre et al., 2002, 2005). We observed that bclA : : Kan spores were also not significantly opsonized by anti-spore IgG when compared to wild-type Ames strain spores (Fig. 4d). Even though BclA remained present on the exosporium of the soaA : : Kan spores, these mutant spores were not significantly opsonized by IgG directed against wild-type ungerminated Ames spores. It is possible that the SoaA protein affected BclA in a way that alters opsonic properties associated with BclA.
To further explore this potential relationship between BclA and SoaA, we performed additional phagocytosis assays. We examined the impact of anti-SoaA antibodies on the phagocytic rates of bclA : : Kan spores (Fig. 4e) as well as the effect of an anti-BclA mAb on the phagocytosis of soaA : : Kan spores (Fig. 4f). Not surprisingly, anti-SoaA antibodies did not opsonize bclA : : Kan spores. These results were predicted, since IgG against whole irradiated spores (already shown to contain antibodies against SoaA, Fig. 3d) also did not opsonize the bclA : : Kan spores. Interestingly, the anti-BclA mAb did opsonize the soaA : : Kan spores at similar levels to those observed with the wild-type spores (Fig. 4f). There are several possible reasons why the mAb opsonizes the soaA : : Kan spores and polyclonal rabbit IgG against irradiated spores does not. In addition to being raised/elicited in two different animal species, the mAb was raised against unfixed purified exosporium and the polyclonal IgG was generated in response to whole irradiated spores. The BclA presentation to the host immune response is likely to be quite different when utilizing these two different methods of antigen preparation. Nonetheless, our results suggest that several spore antigens, including SoaA and BclA, contribute to opsonization with polyclonal antisera.
We also observed that antibodies directed against the SoaA protein could affect wild-type spores (Fig. 4b, c). The observation that the anti-SoaA IgG stimulated phagocytosis of wild-type spores (but not of the soaA : : Kan mutant) (Fig. 4b), like that of anti-whole irradiated spore antibodies (Fig. 4a), supports the hypothesis that the SoaA protein can be found on the surface, at least transiently, and is immunogenic. The observation that antibodies against the small soluble 6 kDa portion of the protein significantly facilitated spore phagocytosis by macrophages argues that this hydrophilic region is a surface-exposed and/or immunodominant segment of the SoaA protein that is recognized by the immune response to gamma-irradiated spores (Fig. 3d).
The apparent release or degradation of SoaA appeared to occur during the process of germination, as discussed above. Conceivably, this might have influenced the early events in germination or spore trafficking in vivo, such as those that were involved in the decreased fitness observed in infected guinea pigs (Fig. 5). Specifically, while inactivating the soaA gene did not alter virulence in a statistically significant manner in mice or guinea pigs (data not shown), the soaA : : Kan mutant appeared to be significantly less suited for survival when competed against the wild-type Ames strain within the guinea pig host (Fig. 5). As shown in vitro, the spores of the soaA : : Kan strain retained most resistance phenotypes that were measured in this study, and the vegetative cells grew at similar rates to those of the Ames strain (data not shown). However, the soaA : : Kan spores were observed to have an altered germination phenotype as determined by loss of heat resistance. While this germination phenotype was undetectable by other germination assays, the loss of heat resistance may constitute an alteration of a relatively early stage of spore germination. These data suggest that the mutant spores germinate more readily or more rapidly than wild-type spores. At the very least, the soaA : : Kan spores lose the characteristic heat-sensitivity more rapidly than wild-type spores. However, we hypothesize that this germination phenotype reflects an alteration towards the end of the initial stage I of germination but before the commencement of stage II of germination which involves metabolic activation (Moir et al., 2002). These coinfection data illustrate that the subtle, yet consistently significant, ‘hyper-germination’ phenotype in the soaA : : Kan spores may result in a significant disadvantage when coinfections are initiated with soaA : : Kan spores and wild-type spores. Perhaps the germinating soaA : : Kan spores are more rapidly disposed of by the innate immune system than the wild-type Ames spores. This could theoretically allow a greater percentage of Ames spores to survive the host immune response, eventually leading to the greater amounts of Ames bacilli present in moribund animals. It is important to note that while some fraction of the soaA : : Kan spore population could be more rapidly disposed of, the surviving spores remain capable of initiating a fatal infection when single B. anthracis strains are used to initiate infections.
This and other studies highlight the potential of an immune response generated against spore-specific epitopes. It has been demonstrated directly and indirectly by several groups that, in addition to toxin antigens, spore entities also have a protective role in the immune response to an anthrax infection (Brossier et al., 2002; Cohen et al., 2000; Enkhtuya et al., 2006; Glomski et al., 2007; Hahn et al., 2005; Kudva et al., 2005; Welkos et al., 2001).
While rabbit polyclonal IgG directed against SoaA did not significantly protect mice from challenge with Ames spores when compared to pre-immune IgG (Fig. 6), these studies did offer new insight into heterologous passive protection experiments. Although mice passively immunized with rabbit IgG were modestly protected against a B. anthracis infection, the protection was not specific to antibodies directed against B. anthracis (Fig. 6). Protection was only observed when the mice immunized with rabbit anti-spore IgG were compared to mice given buffer alone and was not significant in comparison to mice immunized with pre-immune rabbit IgG. These findings potentially clarify an ongoing debate about the efficacy of anti-spore antibodies in passive protection studies (Goossens et al., 2007; Enkhtuya et al., 2007). Using their homologous passive protection model, Glomski et al. (2007) showed that anti-spore antibodies are not sufficient to protect mice from an infection. However, Enkhtuya et al. (2006), using their heterologous passive protection model, showed that rabbit antibodies can significantly protect mice from an infection when compared to a buffer control. Our data demonstrated that the passive protection seen with rabbit anti-spore IgG in mice was at least partly non-specific.
A better understanding of spore morphology and spore–host interactions may be critical for establishing improved vaccines and therapies for B. anthracis. To counteract engineered and emerging threats, we must identify and exploit multiple bacterial targets involved in several stages of pathogenesis, including spore infection, bacillary replication and intoxication.
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ACKNOWLEDGEMENTS |
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Edited by: A. Fouet
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Received 22 March 2007;
revised 2 November 2007;
accepted 14 November 2007.
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  K. M. Severson, M. Mallozzi, J. Bozue, S. L. Welkos, C. K. Cote, K. L. Knight, and A. Driks Roles of the Bacillus anthracis Spore Protein ExsK in Exosporium Maturation and Germination J. Bacteriol., December 15, 2009; 191(24): 7587 - 7596. [Abstract] [Full Text] [PDF]
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 S. Mukhopadhyay, A. Akmal, A. C. Stewart, R.-c. Hsia, and T. D. Read Identification of Bacillus anthracis Spore Component Antigens Conserved across Diverse Bacillus cereus sensu lato Strains Mol. Cell. Proteomics, June 1, 2009; 8(6): 1174 - 1191. [Abstract] [Full Text] [PDF]
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), 300 µg anti-spore IgG (
), 300 µg anti-SoaA IgG (
) or 300 µg pre-immune rabbit IgG (
). Five hours later, the mice received