Four Superoxide Dismutases Contribute to Bacillus anthracis Virulence and Provide Spores with Redundant Protection from Oxidative Stress

The Bacillus anthracis genome encodes four superoxide dismutases(SODs), enzymes capable of detoxifying oxygen radicals. Thattwo of these SODs, SOD15 and SODA1, are present in the outermostlayers of the B. anthracis spore is indicated by previous proteomicanalyses of the exosporium. Given the requirement that sporesmust survive interactions with reactive oxygen species generatedby cells such as macrophages during infection, we hypothesizedthat SOD15 and SODA1 protect the spore from oxidative stressand contribute to the pathogenicity of B. anthracis. To testthese theories, we constructed a double-knockout ( ${Delta}$ sod15 ${Delta}$ sodA1)mutant of B. anthracis Sterne strain 34F2 and assessed its lethalityin an A/J mouse intranasal infection model. The 50% lethal doseof the ${Delta}$ sod15 ${Delta}$ sodA1 strain was similar to that of the wild type(34F2), but surprisingly, measurable whole-spore SOD activitywas greater than that in 34F2. A quadruple-knockout strain ( ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2) was then generated, and as anticipated, spore-associatedSOD activity was diminished. Moreover, the quadruple-knockoutstrain, compared to the wild type, was attenuated more than40-fold upon intranasal challenge of mice. Spore resistanceto exogenously generated oxidative stress and to macrophage-mediatedkilling correlated with virulence in A/J mice. Allelic exchangethat restored sod15 and sodA1 to their wild-type state restoredwild-type characteristics. We conclude that SOD molecules withinthe spore afford B. anthracis protection against oxidative stressand enhance the pathogenicity of B. anthracis in the lung. Wealso surmise that the presence of four SOD alleles within thegenome provides functional redundancy for this key enzyme.

INTRODUCTION

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INTRODUCTION

Bacillus anthracis, the etiological agent of anthrax, is a gram-positive,nonmotile, spore-forming, rod-shaped, facultatively anaerobicbacterium. The versatility provided by sporulation and facultativevegetative growth demands that B. anthracis be equipped to protectboth life stages from oxygen radicals, such as those encounteredby spores in the environment and those generated endogenouslyin vegetative cells during the course of aerobic vegetativegrowth. Furthermore, as a pathogen, B. anthracis confronts hostileconditions during infection, such as the oxidative burst ofprofessional phagocytes that take up spores and the variousoxidative environments encountered by vegetative cells. Antioxidantenzymes such as superoxide dismutases (SODs), catalases, andperoxidases are primary defense mechanisms utilized by bacteriafor preventing oxidative damage (49) and are all present inmultiple copies in the B. anthracis genome. Along with the smallacid-soluble proteins that protect spore-encased DNA from oxygenradicals and other forms of stress (46), some or all of theseenzymes may play a role in combating the stresses faced by thetwo distinct life cycles of B. anthracis. Here, we investigatethe contribution made by spore-bound SODs to B. anthracis oxidativestress defenses.

SODs are enzymes that catalyze the dismutation of the reactiveoxygen species (ROS) superoxide anion (O₂^–) to hydrogenperoxide and molecular oxygen (34). These enzymes effectivelyscavenge O₂^– anions before they are able to cause cellulardamage either directly or through the generation of more reactivespecies such as hydroxyl radicals or peroxynitrite (19). SODwas first discovered and characterized in the 1960s by McCordand Fridovich (34). Since their discovery, SODs have been foundin almost all aerobes studied as well as in many anaerobes.SODs have been shown to exhibit high levels of conservationand to fall within three main structural classes based uponmetal specificity: copper-zinc, manganese or iron, or nickel.Whereas the documented distribution of nickel SODs within thebacterial community is thus far relatively limited (36), manybacterial species possess SODs of both Cu-Zn and Mn/Fe classes,perhaps due to specialized roles filled by each class. The factthat superoxide anions do not cross nonpolar lipid membranesand the observation that Cu-Zn SODs localize typically to theperiplasm, whereas Mn/Fe SODs localize to the cytoplasm, suggestthat different SODs have distinct roles in combating exogenouslyand endogenously produced oxygen radicals, respectively (33,48). SODs of each class have been implicated as being contributorsto virulence in multiple pathogens including Salmonella entericaserovar Typhimurium (20, 21), Mycobacterium tuberculosis (18,38), Staphylococcus aureus (29), Streptococcus agalactiae (40),Francisella tularensis (4), Neisseria meningitidis (54), Brucellaabortus (22), and Enterococcus faecalis (51). A common elementin the infectious course of many of these pathogens is thatthey are capable of surviving interactions with ROS-generatingphagocytic cells such as macrophages (2, 12, 39).

The B. anthracis genome contains four genes that encode proteinswith conserved SOD domains, including two with putative manganesespecificity (BAS4177 [sodA1] and BAS5300 [sodA2]), one withputative copper-zinc specificity (BAS4777 [sodC]), and one withlikely iron and/or manganese specificity (BAS1378 [sod15]) (37,41). Amino acid sequence identity between the manganese SODsis approximately 54%, with 75% similarity, whereas comparisonsbetween the manganese-bound SODA1 and the iron-bound SOD15 indicatean identity of 45% and a similarity of 60%. A 135-amino-acidstretch at the N terminus of SOD15 distinguishes the proteinfrom other B. anthracis SODs, and BLAST searches do not yieldclues regarding possible roles for this unique domain beyonda likely helical secondary structure. Interclass comparisonsbetween the Mn/Fe SODs and the Cu-Zn SODC indicate comparativelylittle homology, although short regions of similarity do exist.

Previous studies (31, 47) indicated that two SODs, SOD15 andSODA1, are present within the B. anthracis exosporium. Thisredundancy suggests an important role for SODs in the spore,possibly in defending against the macrophage and thus contributingto B. anthracis virulence. Passalacqua et al. (37) previouslyconstructed single deletions of each of the four SOD genes anddemonstrated that none of the deletions led to a significantattenuation of B. anthracis when DBA/2 mice were infected viathe intratracheal route, although the deletion of sodA1 didlead to reduced growth under conditions of oxidative stressand increased sensitivity to endogenously produced superoxideanion in the vegetative state. That report did not investigateSOD activity on the surface of the spore or sensitivity to oxidativestress among spores made from deletion strains. Recently, studiesof related Bacillus species reported a sensitivity of sporesto high oxidative stress of multiple forms (13, 14, 42, 55).These observations raise the possibility that multiple chromosomallyencoded SODs provide redundant and combinatorial protectionof the spore from oxidative stress and suggest that the protectiverole for SODs might be fully apparent only upon the deletionof multiple sod genes from the Bacillus genome. Because of thispossibility, we constructed B. anthracis Sterne strain 34F2with multiple sod deletions to assess the importance of SODsin B. anthracis pathogenicity. We found that spores of a Bacillusstrain in which all four SOD genes were deleted exhibited enhancedsensitivity to agents of oxidative stress, reduced survivalin macrophages, and attenuation upon intranasal challenge ofmice.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Bacterial strains. B. anthracis Sterne (toxigenic and unencapsulated; Naval MedicalResearch Center, Silver Spring, MD) and sod gene deletion strainslisted in Table 1 were used for in vitro and in vivo experiments.

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TABLE 1. Bacterial strains used or constructed in this study

Preparation of recombinant proteins. Our procedures for the construction of a recombinant Escherichiacoli strain that expressed recombinant SOD15, SODA1, SODC, SODA2,BxpB, or BclA with an N-terminal six-histidine tag and for purificationof that protein by nickel affinity chromatography were describedin detail elsewhere previously (6). Briefly, genomic DNA wasextracted from B. anthracis strains with the Easy-DNA kit (Invitrogen,Carlsbad, CA). NdeI-BamHI or XhoI-BamHI fragments that containedeach gene of interest were amplified from the Sterne genomeby PCRs using the Expand high-fidelity PCR system (Roche Diagnostics,Indianapolis, IN) in a PTC200 Peltier thermal cycler (MJ Research,Bio-Rad, Hercules, CA). Primer sets designed from the flankingsequences of the targeted gene (National Center for BiotechnologyInformation [http://www.ncbi.nlm.nih.gov.lrc1.usuhs.edu/entrez/viewer.fcgi?db=nucleotide&val=AE017225])are listed in Table S1 in the supplemental material. PCR productswere purified with the QIAEX II gel extraction kit (Qiagen,Valencia, CA), and the DNA fragments were ligated into expressionvector pET15b (Novagen, San Diego, CA). The resulting expressionplasmids are listed in Table S2 in the supplemental material.DNA sequences were verified with the ABI Prism Big Dye method(Applied Biosystems, Forest City, CA) by the Biomedical InstrumentationCenter at the Uniformed Services University, and recombinantplasmids were then transformed into E. coli BL21(DE3)(pLysS)according to the pET system manual (Novagen, San Diego, CA).His-tagged proteins were expressed from transformants and subjectedto His-Trap nickel affinity column chromatography with the Aktafast protein liquid chromatography system (GE Healthcare, Piscataway,NJ).

Immune serum and antibody preparation. Polyclonal antibodies were generated against each recombinantSOD by methods previously described (6). Briefly, rabbits werevaccinated monthly with 50 µg of purified recombinantprotein in Freund's complete adjuvant for the first inoculationand Freund's incomplete adjuvant for all subsequent immunizations.Production bleeds were taken and analyzed for specific antibodiesbeginning at month 5, with final bleeds taken at month 12. ImmunoglobulinG (IgG) was purified from each final serum bleed by passageof the sample over a protein G column (Pierce Biotechnology,Rockford, IL). Protein concentrations were determined by a microtiterBCA assay (Pierce Biotechnology, Rockford, IL).

Construction of the SOD knockout and restored strains. The construction of the ${Delta}$ bclA deletion strain was described previously(6). Deletion constructs of the sod15, sodA1, sodC, and sodA2genes that were suitable for allelic exchange were created bycloning two NotI-XmaI DNA fragments that contained locus-specificflanking regions into the NotI site of pBKJ236 (27). These fragmentswere created by PCR with primers listed in Table S1 in the supplementalmaterial. Ligation of the two fragments created a precise deletionof the respective open reading frame from the start to the stopcodon, inclusively, with an XmaI site generated at the siteof the locus. A complete list of plasmid constructs createdand used in this study is provided in Table S2 in the supplementalmaterial. The resulting constructs were used to create sod nullmutants in B. anthracis Sterne strain 34F2 by an allelic exchangeprocedure described previously (27). In brief, the pBKJ236-basedplasmid constructs were transferred into strain 34F2 by conjugation,and integrants were isolated by a shift to a replication-nonpermissivetemperature followed by growth at the replication-nonpermissivetemperature with continuous selection for erythromycin resistance.A second plasmid, pSS4332, was then introduced into each integrantby conjugative transfer and selection for kanamycin resistance.Plasmid pSS4332, which represents a modification of previouslypublished methods (27) and performs the functions of the previouslydescribed pBKJ233, mediates cleavage within the chromosomallyinserted pBKJ236-derived construct, an event that stimulatesrecombination with a loss of the integrated plasmid and genereplacement in a portion of the erythromycin-sensitive candidates.The resulting knockout strains are summarized in Table 1. Theabsence of the appropriate SOD loci in the various knockoutconstructs was demonstrated by PCR (as shown in Fig. 2) withprimers listed in Table S1 in the supplemental material. Theseprimers were designed to bind to sequences flanking the regionincluded in each deletion construct described above.

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FIG. 2. Deletion of the B. anthracis SOD genes. PCRs show the deletion and restoration of genes from the sod15 (BAS1378), sodA1 (BAS4177), sodC (BAS4777), and sodA2 (BAS5300) loci. Lower (larger) bands represent intact loci, whereas higher (smaller) bands depict deleted loci in which the gene of interest is deleted and flanking regions are left intact. Primers were designed to bind to sites outside the flanking regions of the targeted loci. Primers used and sizes of PCR fragments generated in these reactions are provided in Table S1 in the supplemental material. Strain shorthand reflects the affected loci of the strain. For example, the ${Delta}$ 15 ${Delta}$ A1 strain possesses deletions (smaller PCR products) at sod15 and sodA1 and wild-type (WT) loci (larger PCR products) at sodA2 and sodC, whereas the Res15,A1 strain constructed from the ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 background possesses restored loci at sod15 and sodA1 and deletions at sodA2 and sodC. KO, knockout.

Strains hereafter referred to as "restorants" were constructedwith the 34F2 ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 quadruple-knockout strainas the parent and represent the restoration of sod genes byallelic exchange. Constructs of the intact sod15 and sodA1 locisuitable for allelic exchange were created by the generationof DNA fragments that contained the intact locus, inclusiveof flanking regions, from B. anthracis Sterne strain 34F2 byPCR with primers listed in Table S1 in the supplemental material.Each PCR-amplified fragment was then cloned into the NotI siteof pBKJ236. The allelic exchange was then conducted in two sequentialsteps as described above for the generation of knockout strains.The restoration of the two sod loci was demonstrated by PCR(shown in Fig. 2) with primers listed in Table S1 in the supplementalmaterial.

Generation and purification of B. anthracis spores. Single colonies of B. anthracis Sterne strain 34F2 or mutantstrains were taken from brain heart infusion (BHI) agar platesand inoculated into BHI broth for culture overnight at 37°C.Each culture was spread onto modified germination (G) medium(30) agar plates [0.2% yeast extract, 0.2% (NH₄)₂SO₄, 1.5% Bactoagar, 0.0025% CaCl₂ dihydrate, 0.05% K₂HPO₄, 0.02% MgSO₄ heptahydrate,0.005% MnSO₄ quatrahydrate, 0.0005% ZnSO₄ dihydrate, 0.0005%CuSO₄ pentahydrate, 0.00005% FeSO₄ heptahydrate]. The plateswere incubated at 30°C for 10 to 14 days in the dark. Coloniesscraped from the surface of the agar were resuspended in distilledwater, washed twice in distilled water, and heat treated at65°C for 1 h to kill any viable vegetative cells. Purificationof spores was done with 58% (vol/vol) Renografin (Renocal-76diluted in distilled water; Bracco Diagnostics, Princeton, NJ).Spores were layered onto 58% Renografin and centrifuged at 6,000x g for 60 min in a swinging bucket rotor. The sedimented sporeswere washed twice with distilled water. After the final sedimentation,the spores were resuspended in distilled water to yield a finalconcentration of 10⁹ to 10¹⁰ CFU/ml, as determined by vegetativeoutgrowth on BHI plates.

Preparation of spore surface protein extract (SSPE). Analysis of the proteins contained within the exosporium wasachieved by the removal of the exosporium from whole sporesvia chemical extraction according to a method described previously(1). Briefly, 0.5 ml of heat-activated spores (10⁹ to 10¹⁰ CFU/ml)was pelleted at 7,000 x g for 15 min at 4°C. Spores werewashed twice with water and resuspended in 0.5 ml of extractionbuffer that contained 0.1 M dithiothreitol, 0.1 M NaCl, and0.5% sodium dodecyl sulfate (pH 10). Samples were incubatedin a 37°C shaking water bath for 2.5 h and pelleted, andthe resulting supernatant was collected. Supernatants were thenfiltered through a 0.2-µm Corning filter and dialyzedagainst phosphate-buffered saline (PBS).

Western blot analysis of SSPE. The presence of individual SODs within SSPE generated from variousB. anthracis strains was detected by Western blot analysis.Twenty microliters of SSPE was loaded onto and separated on4 to 20% Tris-glycine sodium dodecyl sulfate-polyacrylamidegels (Invitrogen, Carlsbad, CA), transferred onto Optitran BA-S83 reinforced nitrocellulose (Whatman GmbH, Dassel, Germany),and blocked overnight at 4°C in PBST (PBS with 0.5% Tween20) with 5% nonfat dry milk. Purified His-tagged recombinantproteins were used as positive controls for antibody recognition,with 80 ng of SOD15, 4 ng of SODA1, 100 ng of SODA2, 25 ng ofSODC, or 100 ng of BclA loaded where appropriate. The nitrocellulosewas then incubated with polyclonal anti-spore protein IgG diluted1:500 in PBST with 5% nonfat dry milk for 1 h at room temperature.Following three 5-min washes at room temperature with PBST,the nitrocellulose was incubated for 1 h at room temperaturewith goat anti-rabbit IgG conjugated to horseradish peroxidase(Bio-Rad, Hercules, CA) diluted 1:10,000 in PBST. Blots werethen washed three times for 5 min each in PBST and developedwith Lumigen PS-3 Acridan (GE Healthcare UK, Buchinghamshire,United Kingdom) and Kodak (Rochester, NY) BioMax XAR film.

Characterization of B. anthracis sod mutants and restored strains. Vegetative growth patterns of the parent B. anthracis Sternestrain, knockout strains, and restorant strains were determinedby the inoculation of BHI agar with cultures grown overnightand measurement of the optical density at 600 nm (OD₆₀₀) atvarious times over 8 to 10 h. For all strains, starting inocularegistered an OD₆₀₀ of between 0.065 and 0.070, values thatcorresponded to between 1.00 x 10⁷ and 1.25 x 10⁷ CFU/ml. Thesedata are available in Table S3 in the supplemental material.The growth rate of each strain was measured in three independentexperiments. Alternatively, 5 x 10⁸ heat-inactivated sporesof each strain were inoculated into 73 ml of preheated modifiedG medium in a 250-ml Erlenmeyer flask. This inoculation yieldeda starting OD₆₀₀ of between 0.040 and 0.051, readings that correspondedto between 3.11 x 10⁶ and 3.90 x 10⁶ CFU/ml. These data arealso presented in Table S3 in the supplemental material. Thecultures were then grown at 37°C with shaking at 250 rpm,and the OD₆₀₀ was measured at various times over 8 to 10 h.The growth rate of each strain was measured in three independentexperiments. Germination efficiencies of the strains were comparedby culturing spores in 5% BHI broth, taking samples from thebroth cultures at various times, and heat inactivating the vegetativecells at 68°C for 1 h, as previously described (6). Theheat-treated broth cultures were then subcultured onto BHI agarto enumerate the residual heat-resistant spores. The declinein heat-resistant CFU over time was tested for each strain inthree independent experiments. Within each experiment, threesamples of each strain were heat treated and counted, and thegeometric mean CFU for like samples was determined. For eachof these assays, error bars represent variations among threelike samples at each time point ±1 standard deviation.

Measurement of SOD activity in vitro. The SOD activity of each of the four recombinant SODs or recombinantspores was determined with the SOD Assay Kit-WST (Dojindo MolecularTechnologies, Gaithersburg, MD) (16). In this assay, superoxideanions generated from the oxidation of xanthine by xanthineoxidase (XO) react with colorless water-soluble tetrazoliumsalt (WST) to generate a yellow compound, WST-1 formazan (observedspectrophotometrically at 450 nm). This reaction is inhibitedin a competitive fashion when superoxide anions are degradedby active SOD. We measured SOD activity as the percent inhibitionof WST-1 formazan formation when 2 µg of recombinant SODprotein or between 1.0 x 10⁸ and 1.0 x 10⁹ heat-activated sporesin a total volume of 30 µl were mixed with 200 µlof WST working solution (containing WST and xanthine) and 20µl of XO working solution. Samples were incubated for20 min at 37°C. To measure the SOD activity associated withwhole spores, samples were clarified by centrifugation at 13,200x g for 5 min at 4°C, and the OD₄₅₀ of the supernatant wasdetermined. The percent inhibition of the reaction was calculatedby comparing the values to positive (no oxidation of WST inthe absence of XO) and negative (no inhibition of WST oxidationin the absence of sample or the use of an irrelevant proteinsample) controls. Percent inhibition values were then convertedto units of activity from a standard curve (Fig. 1A) that wasgenerated from a recombinant SOD of known specific activity(manganese-containing E. coli SOD; Sigma-Aldrich, St. Louis,MO). For this mathematical conversion, an equation that describesthe steady-state region of the standard curve (i.e., the slopeof the dashed line depicted in Fig. 1A) was used. Results representthe means of three assays, and the error bars indicate ±1standard deviation.

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FIG. 1. Demonstration of spore-associated SOD activity. (A) SOD activity was measured spectrophotometrically with the Dojindo SOD Activity Kit-WST. Activity in that kit is expressed as the percent inhibition of the XO-mediated conversion of colorless WST-1 to yellow WST formazan, as described in Materials and Methods. We then used an E. coli manganese SOD (eSOD) of known specific activity to generate a standard curve from which to convert percent inhibition to units of SOD activity. For this mathematical conversion, an equation that describes the steady-state region of the standard curve (i.e., the slope of the dashed line depicted in the figure) was used and is presented. (B) Spore-associated SOD activity was assessed using 2.5 x 10⁹ or 2.5 x 10⁸ 34F2 spores. E. coli manganese SOD of known specific activity was used as a positive control. An irrelevant B. anthracis exosporium protein (2 µg of His-tagged, purified BxpB) was included as a negative control. (C) 34F2 and ${Delta}$ bclA spore-bound SOD activity (percent inhibition) in the presence of anti-SOD or control (anti-BxpB) antibodies. *, incubation with ${alpha}$ -SOD15 plus ${alpha}$ -SODA1 polyclonal antibodies yielded a statistically significant decline in measurable SOD activity (P = 0.004); #, incubation with ${alpha}$ -SOD15 or ${alpha}$ -SODA1 alone failed to yield a statistically significant decreases (P $≥$ 0.087).

Test of inhibitory effect of anti-SOD antibodies on spore-associated SOD activity. The impact of polyclonal anti-SOD antibodies on spore-associatedSOD activity was evaluated by means of the SOD Assay Kit-WST.For that purpose, samples of 2.5 x 10⁸ heat-activated sporeswere sedimented by centrifugation and resuspended in 50 µlof PBS that contained 100 µg of appropriate anti-SOD polyclonalIgG. Spore-antibody mixtures were incubated for 24 h at 4°Cwhile shaking. Spores were then washed once in PBS to removeunbound antibody and suspended in a final volume of 30 µl.SOD activity was determined with the kit as described above.

Assessment of sensitivity of vegetative bacilli to endogenously generated oxidative stress. The SOD activity of vegetative bacilli was determined by anagar plate assay as previously described (37), with modifications.Briefly, 200-µl aliquots of 2.5 x 10⁷ CFU/ml heat-activatedspores were added to 2.8 ml of 0.7% sterile soft BHI agar. The3-ml suspension was then spread onto BHI plates. Sterile paperdisks (6 mm in diameter) permeated with 10 µl of 5% paraquat(PST-740; Ultra Scientific) dissolved in water were placed atopthe solidified soft agar layer. Paraquat is a commercially usedherbicide which interacts with the redox pathway of respiringcells and undergoes cyclic reduction-oxidation, a process whichgenerates superoxide anion as a by-product. Given that superoxideanions do not diffuse across lipid membranes, the use of paraquatenables an assessment of the level of SOD protection availablewithin vegetative bacilli. The plates were then incubated overnightat 37°C. The sensitivity of bacilli to endogenous superoxideanions generated from paraquat was measured as the area (inmm²) of growth inhibition around the disk. For each experiment,10 disks (2 disks per plate) were used.

Evaluation of spore sensitivity to exogenously generated oxidative stress. Spore survival in the presence of exogenously generated superoxideanion was tested by methods described previously (22). Briefly,heat-activated spores were washed twice with PBS (pH 7.5) andresuspended at a final concentration of 5 x 10⁶ CFU/ml in PBSwith xanthine at a final concentration of 1 mM, along with 0.5units of XO and 1,000 units of catalase dissolved in 50 mM potassiumphosphate (pH 7.5). Spores were incubated at 37°C, and sporeviability was assessed at 0, 15, 30, and 60 min by colony countsof serial 10-fold dilutions of samples plated onto BHI agar.Results were expressed as the percent spore survival measuredby comparing spore counts at a given time (t_x) with those presentat the initial time (t₀).

Macrophage infection model. The interaction of spores with macrophages of the RAW264.7 cellline (American Type Culture Collection [ATCC], Rockville, MD)was studied by methods detailed previously (28, 53), with modifications.Briefly, the cell line was grown in Dulbecco's minimal essentialmedium (DMEM) with 10% fetal bovine serum (Lonza, Walkersville,MD). Seed cultures were subcultured onto six-well plates andincubated at 37°C in air with 5% CO₂ for 3 to 4 days untilcells reached a density of (0.8 to 1.2) x 10⁶ cells/well. Cellswere washed three times with PBS and infected at 5 x 10⁶ CFU/wellwith spores suspended in serum-free DMEM that contained 100ng/ml phorbol 12-myristate 13-acetate (PMA) (8). Cells wereincubated at 37°C in air with 5% CO₂ for 45 min. Unphagocytosedspores were then removed by washing the wells three times withPBS. Serum-free DMEM that contained 100 ng/ml PMA and 50 µg/mlgentamicin was added to each well, and cells were incubatedat 37°C in air with 5% CO₂ for 45 min to kill extracellularbacteria. The cells were then washed three times with PBS, andfresh serum-free DMEM was added. Cells were incubated at 37°Cin air with 5% CO₂ for 0, 2, 4, and 6 h, after which bacterialviability was assessed as follows. At each harvest time, cellswere washed three times with PBS and incubated for 5 min in1 ml of 0.01% bovine serum albumin in water to lyse macrophages.Wells were scraped with a sterile rubber syringe stopper andpipetted into tubes. Lysates were heated at 65°C for 1 hto kill germinated spores before 10-fold serial dilutions wereprepared for colony counts on trypticase soy agar plates. Theviability of the bacteria was expressed as the percent survivalat a given time point (t_x) compared to that for a sample takenat an initial time point (t₀).

Mouse challenge model. The 50% lethal doses (LD₅₀s) of 34F2, mutant strain, and restorantstrain spores were determined for both the intranasal and subcutaneousroutes of infection. For intranasal spore challenge, mice weresedated with isoflurane with an XGI-8 gas anesthesia system(Xenogen Corporation). Heat-activated spore suspensions of variousconcentrations (see below) in 25 µl were placed onto thenares of anesthetized mice, and the mice were held upright untilthe suspension was inhaled (45). Treatment groups of 10 micereceived doses of spores that ranged from $~$ 10³ to 10⁶ 34F2 sporesand $~$ 10³ to 10⁷ mutant and restorant strain spores. For subcutaneousspore challenge, 200 µl of each dose of heat-activatedspores in suspension was delivered through a tuberculin syringefitted with a 29-gauge needle into the loose skin beneath theright foreleg of the mouse. Treatment groups of 10 mice for34F2 challenge and 5 mice for mutant and restorant strain challengesreceived doses that ranged from $~$ 10² to 10⁵ spores.

Statistical evaluations. The vegetative growth curves of the wild-type and mutant strainswere generated by plotting the average outcomes (OD₆₀₀) of threeexperiments per strain. SOD activity differences were analyzedby one-way analysis of variance (ANOVA) followed by Tukey'spairwise post hoc comparisons. Differences in spore germinationrates or percent survival of spores exposed to exogenously generatedoxygen radicals or macrophages were appraised by repeated-measuresANOVA followed by one-way ANOVA and Tukey's pairwise post hoccomparisons. The LD₅₀ of each selected strain used in theseexperiments, along with 95% confidence limits, was calculatedby probit analysis. Differences in survival and mean time todeath (MTD) for an intranasal challenge at 10⁶ spores were calculatedby Fisher's exact test and Kaplan-Meier analysis, respectively.

RESULTS

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RESULTS

Detection of SOD activity within the spore structure. Heat-inactivated whole spores of B. anthracis Sterne strain34F2 were tested for the presence of SOD activity by a commerciallyavailable in vitro assay that spectrophotometrically measuresthe dismutation of superoxide anions. A standard curve (Fig.1A) generated using a well-characterized E. coli SOD of knownspecific activity allowed the values generated by this assayto be converted into units of enzymatic activity (16). Usingthese methods, spore-associated SOD activity was detectable(Fig. 1B), as was activity associated with recombinant formsof each of the four SODs encoded by the B. anthracis genome(data not shown). While the expression of these recombinantproteins in an artificial system makes it difficult to ensureproper folding and metallation and therefore to make statementsabout relative levels of activity, the confirmation of somelevel of activity above background in the presence of each recombinantform indicates that each protein is in fact a functional SOD.In combination with the detection of spore-associated SOD activityand previous data that localized SODs to the B. anthracis spore(31, 47), these data support the assertion that SOD15 and SODA1are active within the spore.

We next tested whether polyclonal IgG raised against SOD15 andSODA1 could inhibit the activity of SODs on the surface of wild-type34F2 spores and spores of an isogenic bclA mutant from whichthe outer hair-like nap was no longer expressed (see reference6 for a description of the properties of that strain). Althoughthese anti-SOD antibodies had no effect on SOD activity foundon the surface of wild-type 34F2 spores, most likely due tothe barrier effect of BclA on the outer surface of the exosporium,they were capable of reducing activity in the presence of ${Delta}$ bclAspores (Fig. 1C). Moreover, the inhibitory effect of each individualanti-SOD antibody was additive, as indicated by the greatertotal inhibition noted upon the simultaneous incubation of ${Delta}$ bclAspores with both anti-SOD15 and anti-SODA1 IgG. These effectswere specific, since antibodies against BxpB (a major componentof the exosporium exposed in the absence of BclA) (11) did notaffect spore-associated SOD activity. These data agree withenzyme-linked immunosorbent assay and immunoelectron microscopyresults indicating that anti-SOD antibodies bind specificallyto the surface of whole spores in the absence, but not in thepresence, of BclA (11) and suggest that the SOD activity associatedwith the spore is attributable primarily to the spore-boundSOD protein that is localized to the outer layers of the sporeand is available for recognition and inhibition.

A B. anthracis ${Delta}$ sod15 ${Delta}$ sodA1 mutant is unattenuated in an A/J mouse model. To examine the contribution of spore-associated SOD activityto the virulence of B. anthracis, a ${Delta}$ sod15 ${Delta}$ sodA1 double mutantwith markerless deletions of sod15 and sodA1 was generated viahomologous recombination by a modified version of a previouslydescribed method (27) (Fig. 2). This technique replaced theopen reading frame in question with an XmaI site (CCCGGG) andwould not be expected to exert polarity on downstream genes.Mutants were compared to the wild type for defects in growthfrom either cultures grown overnight or heat-inactivated spores(Fig. 3A and B). The ${Delta}$ sod15 ${Delta}$ sodA1 mutant replicated like wild-typestrain 34F2 from a culture grown overnight but showed a slightdelay in vegetative cell outgrowth from spores. We speculatedthat this delay in bacillus growth from ${Delta}$ sod15 ${Delta}$ sodA1 spores mightbe attributable to a delay in the germination of those spores.We then tested this hypothesis and found that the ${Delta}$ sod15 ${Delta}$ sodA1mutant spores did in fact germinate more slowly than did 34F2spores (Fig. 3C). Additionally, the growth of the ${Delta}$ sod15 ${Delta}$ sodA1mutant was more sensitive to oxidative stress, as shown by theincreased zones of inhibition surrounding disks impregnatedwith the redox cycling compound paraquat compared to that for34F2 (Fig. 4A). This increased sensitivity of vegetative cellsto endogenously produced superoxide anion is consistent withthe idea that SODA1 is the predominant protective enzyme duringaerobic growth, as was previously reported (37). However, sporesmade from the ${Delta}$ sod15 ${Delta}$ sodA1 mutant unexpectedly showed increasedlevels of SOD activity compared to levels for 34F2 spores (Fig.4B). Analysis of single ${Delta}$ sod15 and ${Delta}$ sodA1 spores suggested thatthe increase in activity was attributable primarily to the deletionof sod15, as ${Delta}$ sod15 showed an increase in activity similar tothat of the double knockout, whereas the ${Delta}$ sodA1 strain had levelsof activity that resembled those of the wild type (data notshown). Evaluation of the virulence of the ${Delta}$ sod15 ${Delta}$ sodA1 mutantin the Sterne strain-sensitive A/J mouse model indicated noattenuation compared to the wild type as assessed by both intranasaland subcutaneous LD₅₀ values for spore-challenged A/J mice (Table2 and Fig. 5).

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FIG. 3. Growth and germination of B. anthracis wild-type and mutant strains. (A) Vegetative growth of the wild-type Sterne strain and SOD deletion mutants in BHI broth over time after inoculation with cultures grown overnight. (B) Vegetative outgrowth of the wild-type Sterne strain and SOD deletion mutants in modified G broth over time after inoculation with 5 x 10⁸ heat-activated spores. (C) Kinetics of germination of spores from the parent and mutant strains. Spores were inoculated in 5% BHI broth and grown at 37°C and 225 rpm, and samples were heat treated to inactivate vegetative cells at the time points shown. Colony counts following heat treatment represent the numbers of heat-resistant spores. Percent germination at test points (t_x) indicate the numbers of spores that germinated at each time point relative to the spore count at the starting point (t₀). *, the levels of germination of ${Delta}$ sod15 ${Delta}$ sodA1 and ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 spores were statistically lower than those of the wild type at each time point (P < 0.001); **, the level of Res15,A1 germination was lower than that of the wild type at the 5-min (P < 0.001), 10-min (P = 0.002), and 30-min (P = 0.007) time points, but by 60 min, the differences were not significant (P = 0.10).

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FIG. 4. Comparison of SOD activities for the wild-type Sterne strain and mutants. (A) A total of 2.5 x 10⁷ heat-activated spores were mixed with soft agar and spread onto BHI agar, and plates were overlaid with 6-mm disks soaked with paraquat. After overnight incubation of the plates at 37°C, zones of inhibition of bacterial growth around the disk were calculated as the area (mm²) of the disk in which bacterial growth was interrupted (n = 10 disks per strain per experiment). *, zones of inhibition were higher than those for the wild type (P < 0.001). (B) Spore-associated SOD activity from 2.5 x 10⁸ heat-activated spores was measured. *, SOD activity was higher than that of wild-type spores (P < 0.001); **, SOD activity was lower than that of wild-type spores (P < 0.001).

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TABLE 2. LD₅₀ and MTD for intranasal and subcutaneous infections of A/J mice with 34F2 and mutant strains^a

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FIG. 5. Percent survival of A/J mice challenged intranasally with 10⁶ spores of 34F2, ${Delta}$ sod mutant, and sod restorant strains. Treatment groups of 10 mice received 25 µl of a $~$ 4 x 10⁷-CFU/ml heat-activated spore suspension (1 x 10⁶ CFU). Spores were placed onto the nares of anesthetized mice, and the mice were held upright until the suspension was inhaled. Survival of the mice was monitored for a period of 14 days. MTD was significantly different for ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 spores (6.0 days) compared to that for wild-type (WT) spores (3.80 days) (P < 0.005).

Demonstration of functional redundancy among SOD genes in the B. anthracis genome. Western blot analyses of the SSPEs taken from the 34F2 and ${Delta}$ sod15 ${Delta}$ sodA1 strains indicated that upon the deletion of sod15 andsodA1, at least one of the remaining SODs (SODA2) encoded bythe B. anthracis genome was incorporated into the spore (Fig.6). To better understand the nature of the compensation in spore-associatedSOD activity seen with ${Delta}$ sod15 ${Delta}$ sodA1, triple- and quadruple-deletionmutations were generated from the ${Delta}$ sod15 ${Delta}$ sodA1 mutant background(Fig. 2). Analysis of the various deletion strains indicatedthat whereas the deletion of sod15 and sodA1 results in theincorporation of SODA2 into the spore, SODC is incorporatedat a detectable level only when all three other sod genes havebeen (Fig. 6). Further analysis demonstrated that each genotypepossessed a different amount of spore-bound SOD activity (Fig.4B). Only upon the removal of all four sod genes from the genomewas all detectable SOD cleared from the SSPE (Fig. 6), witha corresponding complete elimination of spore-associated SODactivity (Fig. 4B). In addition to being without spore-boundSOD activity, the ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 strain displayed adelay in spore germination as well as a delay in vegetativeoutgrowth from both heat-inactivated spores and cultures grownovernight (Fig. 3). Furthermore, vegetative bacilli of the quadruple-knockoutstrain showed greater sensitivity to paraquat than did 34F2bacilli (Fig. 5A), although the increase in the area of inhibitionbeyond that of ${Delta}$ sod15 ${Delta}$ sodA1 was not statistically significant.

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FIG. 6. Detection of individual SODs within spore extracts by Western blot analysis. Shown are data for incubations of SSPEs prepared from 34F2, mutant, or restorant strain spores with anti-SOD polyclonal IgG. Recombinant proteins were included as controls for antibody recognition (see Materials and Methods).

Attenuation of the ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 strain in the mouse model of intranasal infection. Upon the successful elimination of all spore-bound SOD activity,we attempted to assess the importance of SODs in B. anthracisvirulence by challenging A/J mice with ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2spores (45). Mice were infected by both the subcutaneous andintranasal routes to examine possible differences in the relativeimportances of oxidative stress resistance for each infectiousroute (Table 2). Subcutaneous infection revealed a nonsignificantdifference in LD₅₀s between 34F2 (1.13 x 10³ CFU) and the ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 strain (5.04 x 10³ CFU) of less than fivefold,whereas intranasal challenge showed a variation between 34F2(5.57 x 10⁴ CFU) and the ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 strain (2.51x 10⁶ CFU) of more than 40-fold. While the ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 strain appeared to be attenuated by both routes, onlythe difference in LD₅₀s seen by intranasal inoculation was statisticallysignificant (P < 0.005), as assessed by probit analyses.

Enhanced susceptibility of B. anthracis ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 spores to exogenous oxidative stress. The attenuation of the ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 strain in miceis suggestive of a role for spore-associated SODs in B. anthracispathogenesis. The most obvious role for SODs bound within thespore structure would be to protect against sources of oxidativestress during the early stages of infection. To better understandthe relationship between spore-associated SOD activity and resistanceto oxidative stress, spores were subjected to exposure to exogenouslygenerated superoxide anions (Fig. 7). These superoxide anionswere supplied from the oxidation of the compound xanthine byXO. The ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 mutant spores displayed greatersensitivity to oxidative stress than did 34F2 spores, as demonstratedby the decreased percent survival over time (Fig. 7A). The ${Delta}$ sod15 ${Delta}$ sodA1 mutant spores, which previously demonstrated increasedSOD activity compared to that of 34F2, showed a degree of resistanceno different from that of 34F2.

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FIG. 7. Resistance of wild-type Sterne strain and SOD deletion mutant spores to oxidative stress. A sample of 5 x 10⁶ heat-activated spores was incubated with 1 mM xanthine, 0.5 units XO, and 1,000 units of catalase at 37°C. Samples were taken at 0, 15, 30, and 60 min. *, percent survival for ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 spores was less than that for wild-type spores at all time points (P $≤$ 0.023).

Macrophages kill ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 spores more rapidly than they kill 34F2 spores. Given the observed differences in sensitivity to artificiallygenerated exogenous oxidative stress and the potential roleof ROS as contributors to the destruction of B. anthracis inmacrophages, we next investigated the comparative levels ofsensitivity to macrophage killing. 34F2 and sod mutant sporeswere tested for their abilities to survive phagocytosis by RAW264.7 macrophages. As shown in Fig. 8, phagocytosed ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 spores were more sensitive to killing by macrophagesthan either 34F2 or ${Delta}$ sod15 ${Delta}$ sodA1 spores. The increased levelof SOD activity resident in ${Delta}$ sod15 ${Delta}$ sodA1 spores in comparisonto the level in 34F2 spores did not translate into greater survival,as spores of both strains were killed at the same rate.

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FIG. 8. Resistance of wild-type Sterne strain and SOD deletion mutant spores to macrophage killing. A sample of 5 x 10⁶ heat-activated spores was added to (0.8 to 1.2) x 10⁶ RAW264.7 macrophages in serum-free DMEM plus 100 ng/ml PMA. Spore counts were measured at 0, 2, 4, and 6 h by plating onto tryptic soy agar according to procedures described in Materials and Methods. *, percent survival for ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 spores was less than that for wild-type (WT) spores at 2 h (P = 0.03); #, at 4 h, the difference was not statistically significant (P = 0.08).

Restoration of wild-type SOD activity and virulence by restoration of sod15 and sodA1. In order to further demonstrate that the phenotypes associatedwith ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 were directly related to the absenceof SODs in the spore, we next attempted to restore sod15 andsodA1 to the quadruple-knockout strain. The "restorants" weregenerated in a ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 background by the samemarkerless replacement techniques used in the sod gene deletionstrategy (outlined in Materials and Methods). The sod15⁺ sodA1⁺double restorant (Res15,A1) grew as well as did 34F2, eitherfrom cultures grown overnight or from heat-inactivated spores(Fig. 3A and B). The growth kinetics following the inoculationof heat-inactivated spores into growth medium suggested thatany initial delay in germination (Fig. 3C) was overcome at latertime points and did not significantly hinder vegetative outgrowth.Additionally, Res15,A1 demonstrated wild-type levels of SODactivity both in the vegetative bacilli (Fig. 4A) and on thesurface of the spore (Fig. 4B). The LD₅₀ of spores of the doublerestorant introduced both subcutaneously and intranasally intoA/J mice was equivalent to that of 34F2 (Table 2), as were thekinetics of infection after an intranasal inoculum of $~$ 10⁶ spores(Fig. 5). Finally, in vitro assessments of the sensitivity ofRes15,A1 to oxidative stress (Fig. 7) and macrophage killing(Fig. 8) indicated that the restoration of sod15 and sodA1 enabledlevels of resistance similar to that of 34F2.

DISCUSSION

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DISCUSSION

Here, we describe the construction of a series of sod gene deletionsin B. anthracis that allow an examination of the role playedby SODs in the organism's pathogenesis. Proteomic analyses (31,47) of the B. anthracis spore indicated that at least two ofthe four SODs encoded by the bacterial genome were present inthe endospore. These findings raised the possibility of combinatorialprotection from oxidative stress. To test this theory, we constructeda strain that lacked both spore-associated SODs. The ${Delta}$ sod15 ${Delta}$ sodA1mutant showed a decrease in resistance to oxidative stress inthe vegetative form compared to wild-type strain 34F2, findingsthat are consistent with results described previously by Passalacquaet al. (37). However, spores generated from the ${Delta}$ sod15 ${Delta}$ sodA1strain showed an increase in spore-associated SOD activity.This unanticipated compensation for the loss of sod15 and sodA1was accompanied by the appearance of an alternate SOD specieswithin the spore structure. Furthermore, the ${Delta}$ sod15 ${Delta}$ sodA1 strainwas not attenuated in the A/J mouse model. Additional deletionsof the sodA2 and sodC genes produced a ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2quadruple-knockout strain that displayed a loss of SOD activityassociated with the spore. This ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 strainwas attenuated when inoculated intranasally into mice. Additionally,the virulence of the strains correlated with the sensitivityof the organisms to exogenously produced superoxide anion oractivated macrophages. The restoration of sod15 and sodA1 withinthe quadruple-knockout background generated a strain (Res15,A1)that essentially mimicked wild-type phenotypes in all aspectsrelated to SOD activity, oxidative stress resistance, and virulence.

This study was founded on the hypothesis that SODs incorporatedinto the spore structure afford the spore increased resistanceto oxidative stress that translates into enhanced virulencethrough an increased capacity of spores to survive interactionswith ROS-producing phagocytes. The data presented in this paperstrongly support the notion that the protection from oxidativestress afforded by spore-bound SODs is necessary for the organismto achieve peak virulence, at least within the A/J mouse model.However, alternative explanations for the attenuation seen inthe absence of sod genes remain possible. One such explanationinvolves a role for SODs in proper spore formation. Work doneby Henriques and colleagues (25) with the related species Bacillussubtilis showed that the deletion of the primary bacterial SODled directly to a change in the spore ultrastructure. Thoseauthors speculated that such structural changes indicate a rolefor SODs in promoting the appropriate chemistry necessary forproper spore maturation; i.e., the construction of outer sporelayers might be dependent on a local redox environment thatis affected by the presence or absence of SODs. While thoseauthors reported no discernible effects on spore resistanceproperties in the face of these ultrastructural differences,and while recent studies by Passalacqua et al. (37) similarlyfailed to find profound structural changes upon the deletionof any of the SODs encoded by B. anthracis, it is formally possiblethat the deletion of the four sod genes of B. anthracis mayresult in changes to the spore structure relevant to stressresistance. In such a situation, the increased sensitivity demonstratedby ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 spores in the face of exogenous superoxideanion (Fig. 7) and macrophage phagocytosis (Fig. 8) may in factbe an indication of a change in the resistance properties ofthe spores with respect to multiple forms of stress rather thanan increased sensitivity specifically to oxidative stress.

In this investigation, we observed definite changes in bothB. anthracis growth and germination kinetics that appeared tobe directly attributable to the absence of sod genes. Perhapsnot surprisingly, a delay in the vegetative growth rates ofcertain mutants correlated with an increased sensitivity ofthe vegetative bacilli to endogenous oxidative stress. Intermediateknockout and restorant strains that carried sodA1 or sodA2 aloneexhibited a certain degree of resistance to the effects of paraquatcompared to the ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 strain (Fig. 4A) anddisplayed wild-type growth characteristics (Fig. 3A). Conversely,strains that carried sod15 or sodC alone showed little capacityto grow vegetatively in the face of endogenous oxidative stressand had growth rates similar to those of the ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 strain. These differences indicate a specific role forSOD-mediated protection from internal oxidative stress in achievingmaximum growth rates.

Germination was also affected by the SODs available to the organism.Both the ${Delta}$ sod15 ${Delta}$ sodA1 and ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 strains demonstrateddelayed kinetics compared to those of 34F2 (Fig. 3C), althoughthe total extent of germination is ultimately the same for allstrains (data not shown). The restoration of the sod15 and sodA1loci within the quadruple-knockout background restored wild-typegermination. These results are notable in light of a previousstudy (3) that suggested the possibility of an interplay betweenthe local redox environment and optimal germination. The findingsin that study raise the possibility that the attenuation ofour SOD quadruple mutant might be attributable to delayed-germinationphenotypes rather than to the role of SODs in oxidative stressresistance. However, several pieces of evidence suggest thatslower germination cannot fully explain the reduced virulenceof the ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 strain. The fact that the ${Delta}$ sod15 ${Delta}$ sodA1 double-deletion strain exhibited wild-type virulence despiteslower germination kinetics indicates that a retardation inspore germination does not a priori lead to a diminution invirulence. Furthermore, a delay in germination (or growth) capableof causing attenuation would presumably affect pathogenesisregardless of the route of infection. To the contrary, the LD₅₀data (Table 2) indicate a major difference in the degree ofattenuation depending upon the route of infection, with ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 spores possessing a statistically insignificant4.5-fold decrease in virulence via subcutaneous inoculationand more than a 40-fold attenuation via intranasal entry. Thisdramatic difference seems to argue against delayed germination(or growth) as the central determinant of the attenuation of ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 spores and favors the importance of afactor whose role in pathogenicity is site specific. Of note,available information on the effects of germination kineticson virulence is mixed, as certain reports indicated an increasein virulence associated with a delay in germination (35), whereasother reports showed that delayed germination attenuates virulence(32, 45).

Whereas numerous studies indicate the central role of macrophagesin spore germination and dissemination in the pulmonary infectiousprocess (15, 23, 43-45, 53), recent evidence suggests that sporesinoculated subcutaneously are capable of extracellular germinationand vegetative outgrowth independent of macrophages (5, 9, 10).Dormant and/or germinating spores introduced by the pulmonaryroute must survive interactions with ROS-generating alveolarmacrophages, while macrophage-independent germination via thecutaneous route of entry might provide a less oxidatively stressfulmilieu. Under the conditions found in the phagolysosomal compartmentof the alveolar macrophage, the attenuation of a strain whichlacks the ability to resist oxidative stress would likely beexacerbated. When seen in this way, the severe attenuation ofthe sod mutant seen via the intranasal model is explainableby the loss of a capability that cannot be properly exploitedby the host when bacteria invade subcutaneously. Alternatively,given the potential differences in environmental oxygenationfaced by vegetative bacilli following infection via differentroutes, and the growth differences and differing sensitivitiesof the ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 mutant to endogenous oxidativestress, it is conceivable that the attenuation seen upon intranasalinfection is a reflection of the oxidative stress mediated bythe local host environment.

The stress protection afforded by spore-bound SODs could potentiallybenefit either the dormant spore or the newly germinated bacillus.While the in vitro data described in Fig. 7 and 8 measure ungerminatedspores and suggest a real increase in oxidative stress sensitivityamong spores of the ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 strain, it remainsfeasible that the spore is capable of surviving concentrationsof oxygen radicals and other forms of stress encountered invivo without the benefit of SOD. In this situation, the protectionafforded by SODs bound to the spore might become more importantas B. anthracis undergoes germination within the stressful confinesof the phagocytic vacuole. A previous microscopic examinationof germinating spores demonstrated that the outer layers ofthe spore remain present and continue to envelop newly formedvegetative bacillus (24). Furthermore, it has also been shownthat components of the spore are capable of conferring resistanceproperties to phagolysosome-bound organisms even if the twoare no longer tightly associated (52). The greatest benefitof incorporating SODs into the spore may come only after B.anthracis begins to germinate and sheds the protective shieldafforded by the total spore structure.

Available expression data (37) indicate that while sod15 appearsto undergo a sporulation-specific regulation program, sodA1demonstrates a high level of constitutive expression throughoutall phases of the growth cycle. Thus, through distinct regulatorymechanisms, both SODs detectable within 34F2 spores appear toachieve high levels of expression within the mother cell atthe time of sporulation. According to prevailing models of sporeconstruction (7, 17, 26, 50), these high concentrations wouldmake the proteins available for inclusion in the growing endospore.This inclusion may come about through distinct and specificprotein-protein interactions occurring under the overall directionof the spore's critical morphogenetic proteins (17) or by random,nonspecific mechanics in which the highly abundant proteinsare entangled in the rapidly assembling spore. Either mechanismcould explain the increased spore-associated SOD activity inthe ${Delta}$ sod15 ${Delta}$ sodA1 double mutant. In the event that SODs are incorporatedinto the spore via specific protein-protein interaction competencies,the amino acid similarity between SODs might allow for the inclusionof the remaining SODs in spaces normally occupied by SOD15 and/orSODA1. SODA2, which bears far greater similarity to both SODA1and SOD15 than does SODC, would be better suited for such inclusion.An examination of SODs present in the SSPE generated from thevarious knockout constructions in this study (Fig. 6) supportsthis concept; the deletion of sod15 and sodA1 led directly tothe appearance of SODA2 in the spore extract, whereas SODC wasdetectable only after all other SOD species had been deleted.Additionally, while both sodA2 and sodC are expressed throughoutthe vegetative life cycle of 34F2 at relatively low levels (37),one or both genes may undergo increased levels of expressionas part of a general stress response needed to cope with theloss of primary protection against oxidative stress. In thisscenario, increased concentrations of SODs in the mother cellwould likely facilitate greater nonspecific incorporation intothe spore.

For many bacteria, redundancy among key components is importantfor maintaining fitness. Isozymes can provide distinct yet overlappingcapabilities that allow the bacteria to thrive in multiple environmentalconditions. B. anthracis harbors four sod genes, including onegene (sodC) putatively encoding a Cu-Zn SOD and three genesputatively encoding Mn/Fe SODs. While our work and the workof Passalacqua et al. (37) failed to find a critical role forany one SOD in an A/J or DBA/J mouse model, it is also truethat mice (and humans) represent incidental hosts that makeno significant contribution to the evolution and fitness ofB. anthracis. Given the need for B. anthracis to survive a rangeof diverse hosts and environments, it is perhaps not surprisingthat the organism has evolved to retain such a diverse arrayof SODs. At the same time, the maintenance of these four genesmay provide the bacteria with secondary benefits. Functionalredundancy and compensation through homologous replacement aresupported by previous observations of structural proteins inother Bacillus species (17). Regardless of the reason(s) forthe persistence of multiple sod loci in the genome, our datasuggest that the presence of four sod genes in the B. anthracischromosome allows for compensation in the absence of one ormore species. The spore-associated SOD activity (Fig. 4B) andresistance characteristics (Fig. 7B) of intermediate knockoutand restorant strains carrying only a single SOD species, combinedwith the evidence suggesting that all SODs are capable of inclusionin the spore under the appropriate circumstances (Fig. 6), illustratethat each SOD can contribute to the properties of the spore.However, care must be taken in drawing conclusions about therelative importance of any one SOD to spore activity from databased on an evaluation of its activity in isolation becausethe composite level of SOD activity resident in wild-type 34F2spores is likely dependent on several factors. These factorsinclude, but are not limited to, the inherent specific activityof each SOD, synergistic properties between and among SODs,the concentration of each SOD within the mother cell duringsporulation, and competence for the inclusion of available SODproteins into the spore.

In conclusion, this study revealed an important role for SODsin the full virulence of B. anthracis Sterne in the intranasallychallenged A/J mouse model. The contribution of SODs to virulenceappears to reflect the protection against oxidative stress affordedthe spore during the early stages of infection. We theorizethat the functional redundancy created by the existence of foursod genes within the B. anthracis genome ensures that significantattenuation of the bacteria is seen only with the deletion ofall four genes and the loss of spore-associated SOD activity.The sensitivity of spores to macrophages in the absence of SODssuggests the potential therapeutic utility of small molecularinhibitors that would be capable of blocking the activity ofspore-associated SOD molecules. Finally, enhanced attenuationof the ${Delta}$ sod15 ${Delta}$ sodA1 ${Delta}$ sodC ${Delta}$ sodA2 strain seen via intranasal challengecompared to subcutaneous challenge indicates an important rolefor phagocyte-generated ROS in controlling anthrax infectionsin the lung.

ACKNOWLEDGMENTS

We thank Cara Olson for expert statistical analysis, MichaelFlora for primer synthesis and DNA sequencing, and Jill Czarneckifor polyclonal antibody generation.

This work was supported by NIH/NIAID Middle Atlantic RegionalCenter of Excellence grant U54 AI057168 and research funds fromthe U.S. Navy through Uniformed Services University grant G173HS.We also give special thanks to the U.S. Army Long-Term HealthEducation and Training Program for support of R. Cybulski.

The opinions and assertions in this paper are the private viewsof the authors and are not to be construed as official or asreflecting the views of the Department of Defense. This researchwas conducted in compliance with the Animal Welfare Act. Allanimal use protocols were reviewed and approved by the InstitutionalAnimal Care and Use Committee of the Uniformed Services University.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799. Phone: (301) 295-3400. Fax: (301) 295-3773. E-mail: aobrien{at}usuhs.mil

Published ahead of print on 27 October 2008.

Supplemental material for this article may be found at http://iai.asm.org/.

Editor: F. C. Fang

Present address: FBI Laboratory, 2501 Investigation Parkway,Quantico, VA 22135.

REFERENCES

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