Poly-{gamma}-Glutamate Capsule-Degrading Enzyme Treatment Enhances Phagocytosis and Killing of Encapsulated Bacillus anthracis

The poly- ${gamma}$ -D-glutamic acid capsule confers antiphagocytic propertieson Bacillus anthracis and is essential for virulence. In thisstudy, we showed that CapD, a ${gamma}$ -polyglutamic acid depolymeraseencoded on the B. anthracis capsule plasmid, degraded purifiedcapsule and removed the capsule from the surface of anthraxbacilli. Treatment with CapD induced macrophage phagocytosisof encapsulated B. anthracis and enabled human neutrophils tokill encapsulated organisms. A second glutamylase, PghP, a ${gamma}$ -polyglutamicacid hydrolase encoded by Bacillus subtilis bacteriophage ${Phi}$ NIT1,had minimal activity in degrading B. anthracis capsule, no effecton macrophage phagocytosis, and only minimal enhancement ofneutrophil killing. Thus, the levels of both phagocytosis andkilling corresponded to the degree of enzyme-mediated capsuledegradation. The use of enzymes to degrade the capsule and enablephagocytic killing of B. anthracis offers a new approach tothe therapy of anthrax.

INTRODUCTION

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INTRODUCTION

Bacillus anthracis is the causative agent of anthrax, primarilya disease of domesticated and wild herbivores, which is alsolethal to humans. While naturally occurring anthrax infectionof humans is rare, the recent bioterrorist use of letters containingB. anthracis spores to cause disease has focused the attentionof the biomedical community and generated interest in developingimproved countermeasures. The virulence of B. anthracis is dueprimarily to the presence of two antihost exotoxins (lethaland edema toxins) and an antiphagocytic ${gamma}$ -linked poly-D-glutamicacid capsule surrounding the bacillus that are encoded on separateplasmids, pX01 (36) and pX02 (17, 54). Strains of B. anthracislacking capsule have markedly reduced virulence in animal models(40), and such strains have been used successfully for morethan 50 years to prevent anthrax in domesticated animals (51).Similar vaccines have also been used to control anthrax in humansin countries of the former Soviet Union and the People's Republicof China (48). Capsule synthesis is regulated by factors encodedon both of the virulence plasmids pX01 (11, 12, 20, 53) andpX02 (3, 53, 57) and is induced by the presence of serum andcarbon dioxide (26, 35). The capsule is believed to enhancevirulence by its antiphagocytic property. Although the mechanismby which the capsule inhibits phagocytosis is not firmly established,other bacterial capsules are known to inhibit phagocytosis bytheir anionic charge and by shielding potential bacterial surfaceadhesins (44, 45, 58). Similarly, the capsules of B. anthracisand Bacillus subtilis NAFM5 may function as a physical barrierto phage infection (24, 33).

Phagocytic cells have long been known to play an important rolein anthrax pathogenesis (34). Alveolar phagocytes are thoughtto be involved in transporting inhaled spores to draining lymphnodes, where the spores are thought to germinate (42). Macrophages(60) and neutrophils (23, 32) are reported to phagocytize andkill B. anthracis, and macrophages are required for resistanceto infection in mice (7). Recent work suggests that antibodyto the B. anthracis capsule enhances phagocytosis and killingof encapsulated bacilli (6, 46, 59) and that active (6) andpassive (27) vaccination with capsule protects against experimentalinfection. Taken together, these data suggest that methods toincrease the phagocytosis of encapsulated anthrax bacilli maybe valuable in the treatment of anthrax.

The strategy of using microbial enzymes as antibacterial agentshas a long history, including the use of extracts of Bacilluspyocyaneus to treat anthrax (see reference 2 and referencestherein) and of Aspergillus fumigatus to treat tuberculosis(56). Toenniessen (see reference 2 and references therein) firstreported that an encapsulated bacterium, Klebsiella pneumoniae,became unencapsulated when incubated with Bacillus vulgatus,suggesting that the latter organism secreted an enzyme thatdegraded the capsule. Avery and Dubos extended these ideas todemonstrate that microbial enzymes could be used to remove thecapsule from the pneumococcus and successfully treat experimentalinfections (2). Similar approaches have been used in experimentalEscherichia coli infections (37, 38).

Enzymes capable of degrading the capsule of B. anthracis havebeen isolated from dog liver (55) and from B. anthracis (4,52). The B. anthracis capsule depolymerase gene, capD, encodesa capsule-degrading enzyme, CapD, thought to be necessary forvirulence (4, 31, 52) and for anchoring the capsule to the cellwall peptidoglycan (4). Poly- ${gamma}$ -glutamate hydrolase (PghP) isa 25-kDa enzyme encoded by the bacteriophage ${Phi}$ NIT1 that specificallycleaves D- and L-polyglutamic acid, a component of the capsuleproduced by several strains of B. subtilis (24).

In our study, we found that recombinant CapD degraded high-molecular-weightcapsule, removed capsule from the bacterial surface, and facilitatedmacrophage phagocytosis and neutrophil killing of encapsulatedB. anthracis bacilli. PghP had less activity in degrading capsuleand only a minimal effect on phagocytosis and killing. Theseresults may lead to the development of a novel therapy for anthrax.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Bacterial strains and spore preparation. B. anthracis Ames (pX01⁺ pX02⁺) (U.S. Army Medical ResearchInstitute of Infectious Diseases collection) was cultured inbrain heart infusion (BHI) broth (Becton Dickinson and Co.,Sparks, MD) at 37°C with 0.8% sodium bicarbonate and 5%carbon dioxide (CO₂), except as noted. B. anthracis spores weregenerated as previously described (6). B. subtilis natto (U.S.Department of Agriculture) was cultured at room temperatureon nutrient broth yeast extract agar with 20% CO₂.

Purification of CapD and PghP. The open reading frame of capD, excluding the signal sequence(amino acids 1 to 27), was amplified by PCR and cloned intopET15b (EMD Biosciences, San Diego, CA) as an XhoI-HindIII fragment(forward primer, 5'-GTC GCT CGA GTC TTT CAA TAA AAT AAA AGACAG TGT TA-3'; reverse primer, 5'-GCG GCG AAG CTT CTA TTT ATTTGA TTT CCA AGT TCC ATT CTC TCT GCC-3'). The open reading frameof the pghP gene was amplified from ${Phi}$ NIT1 DNA and cloned intopET15b as a BamHI-NdeI fragment (forward primer, 5'-GCG GCGCAT ATG GCA CAA ACA GAC ACA TAT CCA AAT ATT GAA GCA-3'; reverseprimer, 5'-GCG GCG GGA TCC TTA GCC ATA ATA CTC TGC CTC TGC TTCTTT AAT-3'). Recombinant proteins were expressed and purifiedaccording to the manufacturer's instructions.

Capsule degradation. Capsules from B. subtilis and B. anthracis were purified asdescribed previously (6) and digested with 10-fold dilutionsof purified recombinant CapD or PghP for 1 h at 37°C. Eachreaction mixture contained 1.5 µl (4 µg) of capsule,15.5 µl of phosphate-buffered saline (PBS) (pH 7.4), 1µl of 2 mM ZnSO₄, and 2 µl (1 µg) of enzyme.No ZnSO₄ was added to the reaction mixture containing CapD.There was no effect of ZnSO₄ on capsule migration. After thereaction, an equal volume (20 µl) of 2x sodium dodecylsulfate (SDS)-Tricine sample buffer was added to each sample,and the degradation products were analyzed on a 10% SDS-Tricinepolyacrylamide gel. Gels were washed three times for 3 min inwater, fixed in several changes of 10% propanol with 10% formamide,and stained with StainsAll (10 mg/100 ml [Sigma, St. Louis,MO] in 10% propanol with 10% formamide).

Encapsulated B. anthracis Ames bacilli were prepared by culturingspores for 2 h in BHI broth containing 0.8% sodium bicarbonateat 37°C with 5% CO₂. Bacilli were washed with PBS and adjustedto 1 x 10⁸ bacilli/ml in PBS. CapD or PghP was then added toa final concentration of 50 µg/ml, and cells were incubatedfor 20 min at 37°C. Controls were incubated with PBS alone.Bacterial suspensions were mixed with India ink (Becton Dickinsonand Co.) and visualized by phase-contrast microscopy.

Macrophage phagocytosis. RAW264.7 murine macrophages were grown on coverslips as previouslydescribed (6). B. anthracis Ames spores were resuspended inBHI broth plus 0.8% sodium bicarbonate at a concentration of2 x 10⁷ spores/ml and were allowed to incubate at 37°C in5% CO₂ for 90 min without shaking to generate single, encapsulatedbacilli. For each assay condition, duplicate 1-ml aliquots ofthe newly germinated bacilli were centrifuged and resuspendedin 200 µl of Dulbecco's modified Eagle medium (DMEM) containing10% heat-inactivated fetal bovine serum and 0.1 mM MnCl₂, resultingin a concentration of 1 x 10⁸ bacilli/ml. Manganese or zinc,required as a cofactor for PghP activity (24), was includedin all experiments with PghP. Purified CapD or PghP was addedto a final concentration of 20 µg/ml. In some experimentswith PghP, erythromycin was added to a final concentration of0.5 µg/ml in both PghP and PBS control samples. Experimentswere also performed without antibiotics for comparison. Thesuspensions were incubated for 10 min at 37°C and then layeredonto coverslips containing adhered macrophages and allowed toincubate at 37°C in 5% CO₂ for 30 min. In some experiments,opsonization of encapsulated bacilli with a high-titer anticapsuleor normal mouse serum (21) at a 1:20 dilution was carried outfor 30 min prior to layering onto macrophages. After the incubation,coverslips were washed extensively in PBS (pH 7.4) and stainedwith Wright Giemsa stain. Phagocytosis was measured by countingthe number of adherent bacilli on approximately 100 macrophagesfrom each of two coverslips. The phagocytic index was definedas the average number of bacilli that adhered to each macrophage± standard error of the mean.

Human neutrophil bactericidal activity. Human neutrophils were isolated from healthy, unvaccinated volunteersusing Histopaque 1077 and 1119 gradients according to instructionsprovided by the manufacturer (Sigma-Aldrich, St. Louis, MO)or by Ficoll-Hypaque density gradient centrifugation followedby dextran sedimentation (28) and resuspended in DMEM at a concentrationof 2 x 10⁷/ml. Results were the same with neutrophils purifiedby either method. Single, encapsulated B. anthracis bacilliprepared as described above were resuspended in DMEM at 1 x10⁷ bacilli/ml and treated with either PBS or 20 µg/mlof CapD or PghP for 10 min at 37°C. In some experiments,encapsulated bacilli were instead pretreated for 30 min withanticapsule or normal mouse serum at a 1:20 dilution. Neutrophilswere adjusted to a final concentration of 5 x 10⁶ neutrophils/mlin a 1.5-ml microcentrifuge tube in duplicate, and enzyme-treatedbacilli were added to 1 x 10⁵ bacilli/ml for a neutrophil-to-bacillusratio of 50:1, except as noted. Normal, autologous human serum(10%) was included as a source of complement, and enzymes oranticapsule serum was added to maintain a final concentrationof 20 µg/ml and 5%, respectively. The total reaction mixturevolume was 550 µl. An aliquot was immediately removedat time zero to determine CFU by serial dilution in water andplating onto Luria-Bertani agar. The sample was then rotatedat 37°C, and bacterial viability was measured at 2 h. Todetermine the effect of phagocytosis inhibition on CapD-mediatedkilling, cytochalasin D (Sigma-Aldrich) was added to neutrophils(10 µg/ml, 10 min, 37°C) prior to the addition ofbacilli.

Statistics. Phagocytic index comparisons were evaluated for statisticalsignificance by analysis of variance (ANOVA) with Tukey's posthoc tests between groups. ANOVA for phagocytic indices betweengroups was significant (P < 0.0001). Differences in neutrophilkilling between groups over time were evaluated with repeated-measuresANOVA and shown to be statistically significant (P < 0.0001).Differences within a group between time zero and 2 h were testedusing post hoc paired t tests. Differences between groups at2 h were tested using Tukey's post hoc tests. P values for posthoc analysis are indicated in the text.

RESULTS

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RESULTS

Hydrolysis of capsule with purified CapD and PghP. Encapsulated B. anthracis Ames bacilli were treated with recombinantCapD or PghP and visualized by phase-contrast microscopy. Capsuleremoval was observed as early as 5 min after treatment witheither enzyme, and after 20 min, very little capsule was visible(Fig. 1). Treatment with CapD at a concentration as low as 0.035µg/ml was sufficient to visibly decrease the size of thecapsule by India ink staining.

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FIG. 1. Removal of capsule from encapsulated B. anthracis by CapD and PghP. B. anthracis Ames bacilli were grown under conditions to express capsule and treated for 20 min at 37°C with PBS (A), 50 µg/ml CapD (B), or 50 µg/ml PghP (C) as described in Materials and Methods and examined by India ink and phase-contrast microscopy (magnification, x1,000).

To determine the extent of enzyme degradation, capsule purifiedfrom B. anthracis Ames and B. subtilis was digested with CapDor PghP for 1 h at 37°C and examined by SDS-polyacrylamidegel electrophoresis on a 10% SDS-Tricine gel. As seen in Fig.2, the B. anthracis capsule was digested to completion with35 µg/ml of CapD, and activity was clearly detectableat 3.5 µg/ml (lanes 4 to 6). CapD also hydrolyzed theB. subtilis capsule to completion at 35 µg/ml (lane 3).By contrast, PghP had significantly less activity than CapDon the B. anthracis capsule (Fig. 2, compare lanes 10 to 12to lanes 4 to 6) but was more active than CapD on the B. subtiliscapsule, with activity detected at concentrations as low as0.035 µg/ml (lanes 7 to 9).

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FIG. 2. Degradation of capsules from B. anthracis and B. subtilis by CapD and PghP. Capsule purified from B. subtilis (lanes 1 to 3 and 7 to 9) and B. anthracis (lanes 4 to 6 and 10 to 12) were digested with CapD (lanes 1 to 6) or PghP (lanes 7 to 12) and examined by SDS-polyacrylamide gel electrophoresis as described in Materials and Methods. Lanes 1 and 4, 0.35 µg/ml of CapD; lanes 2 and 5, 3.5 µg/ml of CapD; lanes 3 and 6, 35 µg/ml of CapD; lane 7, 0.035 µg/ml of PghP; lanes 8 and 10, 0.35 µg/ml of PghP; lanes 9 and 11, 3.5 µg/ml of PghP; lane 12, 35 µg/ml of PghP.

Phagocytosis of enzyme-treated encapsulated bacilli. We next examined the effect of enzymatic treatment of encapsulatedB. anthracis on phagocytosis by macrophages. Encapsulated B.anthracis bacilli were treated with purified CapD or PghP andthen incubated with RAW264.7 murine macrophages, and the phagocyticindex was determined as described in Materials and Methods.Treatment with CapD resulted in a dramatic increase in phagocytosis.The phagocytic index increased from 1.3 ± 0.2 to 43 ±4.7 (Table 1) (P < 0.0001 [ANOVA]; for CapD versus PBS, P< 0.0001 [Tukey's post hoc test]). PghP treatment did notincrease the phagocytic index above that seen with PBS, althoughin some experiments, a slight effect was observed. The higherlevel of CapD-mediated phagocytosis is consistent with the greaterdegree of degradation of purified B. anthracis capsule observedwith CapD than with PghP (Fig. 2). We observed that capsuleregeneration occurred during incubation in 5% CO₂, which couldreduce the effect of the PghP enzyme due to its relatively lowefficiency of capsule degradation. To counteract this, a bacteriostaticconcentration of erythromycin (0.5 µg/ml) was added toinhibit the regrowth of the capsule. Under these conditions,increased phagocytosis was observed after PghP treatment, withthe phagocytic index increasing 5.6-fold from 1.2 ± 0.3in the PBS control with erythromycin compared to 6.7 ±2.4 in cells treated with PghP and erythromycin (P < 0.0001[ANOVA]; P < 0.0001 for PghP versus PBS [Tukey's post hoctest]) (data not shown). Treatment with erythromycin alone inthe absence of PghP had no effect on the phagocytic index comparedto PBS alone (1.2 ± 0.3 versus 1.3 ± 0.2) (P <0.0001 [ANOVA]; P > 0.1 for PghP versus PBS [Tukey's posthoc test]). There was no effect of erythromycin on CapD-mediatedphagocytosis (data not shown). Adherence of enzyme-treated bacillito macrophages is illustrated in Fig. 3. The extent of phagocytosisobserved after treatment with CapD was similar to that seenwith unencapsulated bacilli that germinated from spores in theabsence of sodium bicarbonate and CO₂ (Table 1) (P < 0.0001[ANOVA]; P < 0.0001 for unencapsulated bacilli versus PBS[Tukey's post hoc test]). Opsonization of encapsulated bacilliwith anticapsule serum resulted in a significant increase inthe phagocytic index to 6.5 ± 0.39 compared to 1.1 ±0.1 with normal mouse serum (P < 0.0001 [ANOVA]; P < 0.0001for anticapsule versus normal serum [Tukey's post hoc test])(data not shown).

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TABLE 1. Enzyme treatment enhances macrophage phagocytosis and neutrophil killing of encapsulated B. anthracis Ames

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FIG. 3. Phagocytosis of encapsulated B. anthracis by RAW264.7 macrophages. Encapsulated B. anthracis Ames bacilli were treated with PBS (A), CapD (20 µg/ml) (B), or PghP (20 µg/ml) (C) and incubated with macrophages as described in Materials and Methods. The PBS and PghP samples contained 0.5 µg/ml of erythromycin.

Enzyme-mediated neutrophil killing of encapsulated bacilli. We then determined the effect of treating bacilli with recombinantCapD or PghP on their killing by human neutrophils. Newly germinated,encapsulated B. anthracis bacilli that were pretreated withenzymes were incubated with neutrophils in the continued presenceof enzymes, and bacterial viability was measured by serial dilutionand plating. Treatment with purified CapD, and to a lesser extentwith PghP, facilitated neutrophil killing of encapsulated B.anthracis (Table 1). CapD treatment resulted in neutrophil killingof >99% of encapsulated bacilli, with viability falling to0.5% (Table 1) (P < 0.0001 [ANOVA]; P < 0.0001 for CapDat time zero versus that at 2 h [paired t test]) of the inputbacilli after 2 h incubation. This extensive degree of killingis similar to that observed with unencapsulated bacilli producedby growth in the absence of sodium bicarbonate and CO₂ (Table1) (P < 0.0001 [ANOVA]; P < 0.0001 for unencapsulatedbacilli at time zero versus that at 2 h [paired t test]). Itwas also similar to the neutrophil killing observed after opsonizationof encapsulated bacilli with anticapsule serum (>99% after2 h incubation) (data not shown). In six other experiments withCapD treatment, killing after 2 h was $≥$ 99%. Similar results wereobserved (>99% killing) at a neutrophil-to-bacillus ratioof 1:1 (Fig. 4). Treatment with PghP resulted in only a moderatedegree of neutrophil killing, with viability at 2 h reducedto 52.5% (Table 1) (P < 0.0001 [ANOVA]; P < 0.0001 forPghP at time zero versus that at 2 h [paired t test]) and 29.4%(P < 0.0001, paired t test) in two experiments compared tothat at time zero. The effect of PghP treatment on bacterialviability was significantly less than that of CapD (Table 1)(P < 0.0001 [ANOVA]; P < 0.0001 for CapD versus PghP [Tukey'spost hoc test]). This again is consistent with the lower activityof PghP compared to CapD in degrading the purified B. anthraciscapsule (Fig. 2). To eliminate the possibility that high-molecular-weightcapsule fragments released by PghP treatment could impair neutrophilkilling, we prepared the supernatant containing released capsulesfrom encapsulated bacilli treated with PghP and added it toneutrophils incubated with unencapsulated bacilli as describedin Materials and Methods. The addition of this supernatant didnot impair the ability of neutrophils to kill unencapsulatedbacilli (data not shown).

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FIG. 4. CapD-mediated killing of B. anthracis Ames by human neutrophils. Human neutrophils (5 x 10⁶ neutrophils/ml) were mixed with encapsulated bacilli (5 x 10⁶ neutrophils/ml) with (A) or without (B) 20 µg/ml of CapD and incubated for 3 h at 37°C on an Eppendorf tube rotator. Cells were then concentrated 10-fold by centrifugation and stained with Wright Giemsa stain (magnification, x1,000). Viability was reduced by CapD treatment to 0.35% after 3 h compared to that at time zero. No killing occurred in the sample (B) incubated without CapD.

In the absence of enzyme treatment, a moderate decrease in bacterialviability was observed after incubation with neutrophils insome experiments, while modest growth occurred in others. Inthe experiment presented in Table 1, killing (28% decrease inCFU) was observed at 2 h (Table 1) (P < 0.0001 [ANOVA]; P= 0.0025 for PBS at time zero versus that at 2 h [paired t test]).The average percent viability at 2 h in the PBS control groupsfrom seven experiments was 98.6 ± 26.7 (standard errorof the mean). There was variability using different donors,with statistically significant (P < 0.0001 [ANOVA]; P <0.05 for PBS at time zero versus that at 2 h [paired t test])killing observed in four experiments, while statistically significantgrowth was seen in the three other experiments. An additionalcontrol showed that encapsulated bacilli treated with CapD alonein complete medium (DMEM containing 10% normal human serum)in the absence of neutrophils were not killed but rather doubledin CFU by 2 h (data not shown). Thus, efficient killing of encapsulatedbacilli required both CapD treatment and neutrophils. Optimalkilling ( $≥$ 99%) occurred in the presence of normal but not heat-inactivatedhuman serum (data not shown), indicating the likely requirementfor complement. Additionally, treatment of neutrophils withcytochalasin D (10 µg/ml) almost completely blocked thekilling of CapD-treated bacilli, with 67.9% survival, comparedto 0.5% with CapD alone (Table 1) (P < 0.0001 [ANOVA]; P< 0.0001 for CapD versus CapD plus cytochalasin D [Tukey'spost hoc test]), indicating that killing was phagocytosis dependent.

In the next experiment, we determined the minimal concentrationof CapD required to mediate neutrophil killing. As shown inFig. 5, CapD treatment enhanced neutrophil killing in a concentration-dependentmanner. The concentration of CapD necessary to facilitate optimalneutrophil killing of encapsulated bacilli by 2 h was approximately1 µg/ml (Fig. 5), and 0.25 µg/ml was sufficientto facilitate the killing of >90% of bacilli.

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FIG. 5. CapD concentration-dependent neutrophil killing. CapD was serially diluted in PBS and used to treat encapsulated B. anthracis Ames bacilli before being mixed with human neutrophils as described in Materials and Methods. The bacterial viability of duplicate samples (means ± standard deviations) was measured at 2 h.

DISCUSSION

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DISCUSSION

During the initial stages of infection by the aerosol route,spores are thought to be engulfed by macrophages or other phagocyticcells and are transported to regional lymph nodes. Some organismsare killed, likely those that germinate (22, 38a, 42, 60), whileothers remain viable and proliferate (43, 47). Germinated sporesand bacilli are thought to eventually escape from the macrophageinto the lymph nodes and then the bloodstream, where they spreadsystemically to most organs and replicate virtually unimpededas short chains of phagocytosis-resistant encapsulated bacilli.Evidence suggests that the toxins (19) and capsule (15) aresynthesized early after germination. The lethal and edema toxinsare thought to interfere with macrophage function, althoughtheir role in intracellular killing and the fate of B. anthracisitself in macrophages remain unclear, with either killing (60),persistence (18), or growth (10) being reported, which is likelyrelated to methodological differences and the use of differentcells and bacterial strains, both encapsulated and unencapsulated.The toxins have also been reported to interfere with neutrophilfunction (1, 13, 23, 39, 61), while capsule synthesis and encapsulationrenders the bacillus resistant to phagocytosis when tested invitro (23, 30, 49) (Table 1), and in infected animals, encapsulatedbacilli are essentially observed only extracellularly (9). Withhost innate immune defenses compromised, the bacteria grow tolevels as high as 10⁸ to 10⁹ CFU/ml in the blood.

We demonstrated in this study that CapD can remove the capsulefrom the surface of encapsulated B. anthracis bacilli and degradethe purified capsule. Most importantly, encapsulated B. anthracisbacilli that are resistant to phagocytosis can be made highlysusceptible to phagocytosis by macrophages and to killing afteringestion by neutrophils by enzymatically removing the capsulewith CapD. While PghP treatment appeared to remove the capsulefrom the surface of bacilli, it had no effect on macrophagephagocytosis by itself and a minimal effect on neutrophil killing(Table 1). PghP was also much less efficient than CapD in degradingthe purified B. anthracis capsule to lower-molecular-weightspecies (Fig. 2). The B. anthracis capsule is composed entirelyof poly-D-glutamic acid, and PghP, in contrast to CapD, mayhave specificity for mixtures of poly-D- and L-glutamic acidsand thus may only inefficiently degrade the pure D form. Thisis reflected in the dramatically greater activity of PghP onthe poly-D- and L-glutamic acid capsule of B. subtilis thanon the poly-D-glutamic acid capsule of B. anthracis (Fig. 2).Indeed, in preliminary experiments, CapD but not PghP was ableto degrade a 30-mer of poly-D-glutamate (data not shown). Theremay be more residual capsule remaining on the bacillus thatis not apparent by India ink microscopy after PghP treatmentcompared to CapD treatment, which may be responsible for thedifferences in phagocytosis and killing. Another possible explanationfor the minimal effect of PghP treatment on neutrophil killingcompared to CapD treatment may be a "trans" effect due to remaininglarge capsule fragments released into the medium by PghP treatment,which could impair neutrophil killing. This possibility waseliminated by showing that the addition of supernatant fromPghP-treated encapsulated bacilli to neutrophils did not impairtheir ability to kill unencapsulated bacilli under these assayconditions.

In the absence of CapD treatment, minimal killing by neutrophilswas noted in some individuals, while no killing occurred inothers. This may be related to the presence of various amountsof cross-reacting anticapsule antibodies in some individualsdue to prior exposure to other bacteria, as polyglutamic acidcapsules are known to be made by Staphylococcus epidermidis(25) and other Bacillus species and organisms (5). Future studieswill be necessary to address this point.

CapD-mediated neutrophil killing appeared to be dependent oncomplement, as it was reduced when heat-inactivated serum wasused, similar to what has long been reported for other bacteria(16, 28, 41), and complement was shown to be necessary for neutrophilkilling of an unencapsulated B. anthracis strain (data not shown).This is consistent with a preliminary report noting less C3binding to encapsulated than to unencapsulated B. anthracisas well as less killing by human blood cells, although few detailswere provided (40a). Thus, CapD-mediated enzymatic removal ofthe capsule may allow for greater complement deposition andsubsequent neutrophil killing. Further studies will be necessaryto confirm this. A lack of capsule on the bacillus surface mayalso render them more susceptible to the products of phagolysosomalfusion, such as reactive oxygen species and cationic peptides.Indeed, it has been reported previously that capsule can blockthe bactericidal activity of neutrophil extracts (23), and wehave observed that purified capsule can interfere with the bactericidalactivity of some cationic peptides including alpha defensinspresent in neutrophils and beta defensins produced by nonphagocyticcells (38b). Thus, in addition to its antiphagocytic property,the capsule may contribute to resistance to intracellular killingand resistance to nonphagocytic innate immune defenses.

Gamma glutamyltranspeptidase, encoded by capD, has been functionallycharacterized as a virulence factor (4, 31, 52). The 55-kDaprotein has depolymerase activity and degrades high-molecular-weightcapsule with the release of small amounts of lower-molecular-weightspecies that have been suggested to contribute to virulence(31). CapD was also reported to be necessary for anchoring thecapsule to the peptidoglycan (4). It is possible, however, thatthe expression of capD may be tightly regulated, as overexpressioncould lead to a high level of depolymerization and a loss ofcapsule from the bacterial surface. A preliminary proteomicanalysis of B. anthracis grown in medium containing bicarbonateand CO₂ revealed that although capD is on a polycistronic operonwith capB, capC, and capA (52), the amount appeared to be significantlylower than those of the other three components (data not shown).This may indicate that capD is differentially regulated comparedto the other components of the operon.

The strategy of targeting B. anthracis bacilli to phagocytesto treat anthrax infection has only recently been explored.The demonstration of protection in animals mediated by anticapsuleantibodies (6, 27) supports a role for phagocytes in combatinganthrax infection by controlling the encapsulated vegetativebacillus. Macrophages have traditionally been viewed as havinga dual role in the disease: as carriers of B. anthracis sporesto the lymph nodes and thus a vehicle for uptake and disseminationafter inhalation (42) and as a factor in contributing to protection(7). The role of macrophages as carriers and sites of germinationof spores in cutaneous infection is less well established. Polymorphonuclearneutrophils are a primary defense mechanism to control manybacterial infections, but depletion experiments have suggestedthat they play a limited role in natural resistance to encapsulatedB. anthracis infection in the susceptible mouse model (8). Thisis consistent with the idea that once the organism becomes encapsulated,the role of neutrophils in natural resistance is diminished.In the present report, we confirmed the resistance of encapsulatedbacilli to phagocytosis and killing. Most importantly, we observedthat treatment with CapD promoted neutrophil bactericidal activitythat resulted in a $≥$ 99% decrease (2 to 3 logs) in vegetativecell viability, suggesting that efficient capsule removal maypotentially lead to severe attenuation of B. anthracis and subsequentclearance from the host. This degree of killing is similar towhat we observed with unencapsulated bacilli and with anticapsuleantibody. Further experiments with infected animals will beneeded to determine whether enzyme treatment will be as effectiveas anticapsule antibodies.

A recent study by Mayer-Scholl et al. reported that human neutrophilskill encapsulated as well as unencapsulated B. anthracis (32).At a neutrophil-to-bacterium ratio of 0.1:1, there was $~$ 45% and $~$ 70% killing of the encapsulated wild-type bacilli compared to $~$ 90 and $~$ 95% killing of the unencapsulated strain after 2 h and3 h, respectively, showing a minimal effect of the capsule.At a ratio of 1:1, there was no difference in killing of theencapsulated versus unencapsulated bacilli, with $~$ 90% of thelatter killed after only 30 min. In contrast, we observed thatencapsulated bacilli were resistant to killing (<30% killingin five of seven experiments), compared to $≥$ 99% killing afterthe removal of capsule by CapD treatment, confirming the antiphagocyticactivity of the capsule reported previously by others (23, 30,49). The $~$ 2- to 3-log killing that we observed is similar towhat occurs with neutrophils incubated in suspension with otherbacteria such as Staphylococcus (28; data not shown) and occurredat neutrophil-to-bacterium ratios of both 50:1 and 1:1. Thedegree of killing that we observed after capsule removal andfor unencapsulated bacilli was at least 1 log greater than whatMayer-Scholl et al. previously observed for unencapsulated bacilli( $≥$ 99% versus $~$ 90 to 95%). However, there are several notable methodologicaldifferences in the neutrophil killing assays that likely explainthe discrepancies. While we used neutrophils in suspension atneutrophil-to-bacterium ratios of 50:1 and 1:1, those authorsused plastic adherent cells that were preactivated before exposureto bacilli centrifuged onto the neutrophils at neutrophil-to-bacteriumratios of 1:1 and 0.1:1. Furthermore, the killing that theyobserved was predominantly extracellular, as it was only modestlyinhibited by cytochalasin D, and electron microscopy showedmost bacilli to be extracellular. This is in contrast to ourresults, where killing was dramatically inhibited by cytochalasinD (Table 1), showing that the killing that we observed dependson phagocytosis, consistent with prior observations on neutrophilbacterial killing in suspension (29).

The development of methods to promote phagocytosis and killingof encapsulated anthrax bacilli may offer an alternative approachfor the treatment of anthrax. In this regard, it has been suggestedthat the inhibition of CapD may result in a loss of capsulefrom the surface and attenuation of the organism (4). The presentresults suggest that enzymes such as CapD, by degrading thecapsule and making the bacillus susceptible to phagocytic killing,may offer a new approach to therapy that would be both effectiveagainst naturally occurring organisms and of particular valueagainst possible antibiotic- and vaccine-resistant strains.

ACKNOWLEDGMENTS

We thank S. Norris for her expert statistical analysis.

The opinions, interpretations, conclusions, and recommendationsare those of the authors and are not necessarily endorsed bythe U.S. Army.

The research described herein was sponsored by the Medical BiologicalDefense Research Program, U.S. Army Medical Research and MaterielCommand, project number A1_X003_04_RD_B.

FOOTNOTES

* Corresponding author. Mailing address: USAMRIID, 1425 Porter Street, Frederick, MD 21702. Phone for Angelo Scorpio: (301) 619-4935. Fax: (301) 619-4299. E-mail: Angelo.Scorpio{at}amedd.army.mil. Phone for Arthur M. Friedlander: (301) 619-7343. Fax: (301) 619-4299. E-mail: Arthur.Friedlander{at}amedd.army.mil.

Published ahead of print on 30 October 2006.

REFERENCES

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