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Infection and Immunity, March 2008, p. 899-906, Vol. 76, No. 3
0019-9567/08/$08.00+0 doi:10.1128/IAI.01176-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
In Vivo Fate and Distribution of Poly-
-D-Glutamic Acid, the Capsular Antigen from Bacillus anthracis
Marjorie D. Sutherland,1,2
Peter Thorkildson,1
Samuel D. Parks,3 and
Thomas R. Kozel1,2*
Department of Microbiology and Immunology,1
Cell and Molecular Biology Program,2
Department of Pathology, University of Nevada School of Medicine, Reno, Nevada 895573
Received 24 August 2007/
Returned for modification 1 October 2007/
Accepted 27 December 2007
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ABSTRACT
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Bacillus anthracis is surrounded by an antiphagocytic capsule composed of poly-
-D-glutamic acid (DPGA). Bacterial and fungal capsular polysaccharides are shed into body fluids in large amounts during infection. The goal of our study was to examine the in vivo fate and distribution of the DPGA capsular polypeptide. Mice were injected via the intravenous route with various amounts of purified DPGA. Blood, urine, and various organs were harvested at different times after treatment. Sites of DPGA accumulation were determined by immunoassay using monoclonal antibodies specific for DPGA. The results showed that the liver and spleen were the primary sites for the accumulation of DPGA. As found in previous studies of capsular polysaccharides, the Kupffer cells of the liver and splenic macrophages were sites for the cellular accumulation of DPGA. Unlike capsular polysaccharides, the hepatic sinusoidal endothelial cells were also sites for DPGA accumulation. DPGA was rapidly cleared from serum and was excreted into the urine. DPGA in the urine showed a reduced molecular size relative to native DPGA. The results indicate that in vivo clearance of the polypeptide capsular antigen of B. anthracis shares several features with the clearance of capsular polysaccharides. Key differences between the in vivo behaviors of DPGA and capsular polysaccharides include the accumulation of DPGA in hepatic sinusoidal endothelial cells and a DPGA clearance rate that was more rapid than the clearance reported for capsular polysaccharides.
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INTRODUCTION
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Most bacterial capsules are composed of polysaccharides. These capsules are essential to the virulence of many pathogenic bacteria, such as Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis as well as the opportunistic yeast Cryptococcus neoformans. The capsules are characteristically antiphagocytic, and antibodies to capsular polysaccharides are protective. Capsular polysaccharides display a repeating epitope structure, have a high molecular weight, and resist degradation in vivo. These properties are characteristic of antigens classified as thymus-independent type 2 (22).
Unlike the capsular polysaccharides that surround most bacteria, the capsule of Bacillus anthracis is a homopolymer of D-glutamic acid residues that are linked by the gamma carboxyl group (poly--D-glutamic acid [DPGA]) (11). The DPGA capsule is essential for virulence (5, 14, 30). Like capsular polysaccharides, DPGA is poorly immunogenic, and the coupling of DPGA to immunogenic protein carriers greatly enhances immunogenicity (15, 24, 25, 29). Finally, as with capsular polysaccharides, antibodies to DPGA are protective in murine models of pulmonary anthrax (2, 15, 18).
Studies of the in vivo behaviors of capsular polysaccharides of S. pneumoniae, H. influenzae type b, and C. neoformans found that capsular polysaccharides accumulate in cells of the reticuloendothelial system and persist for weeks in tissues and serum. Despite the essential role played by DPGA in the virulence of B. anthracis, little is known regarding its in vivo activities. In a first step toward better understanding the virulence properties of DPGA, we explored the tissue trafficking and clearance of DPGA in vivo. The results showed that, like capsular polysaccharides, DPGA accumulates in cells characteristically associated with the reticuloendothelial system. However, large amounts of DPGA also accumulate in the sinusoidal endothelial cells of the liver. In addition, the overall rate of clearance of DPGA from serum and various tissues is relatively rapid compared to that found in previous studies of capsular polysaccharides.
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MATERIALS AND METHODS
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Bacterial culture and DPGA isolation.
B. anthracis Pasteur is maintained by the Nevada State Health Laboratory (Reno, NV) and was originally obtained from the Centers for Disease Control and Prevention (Atlanta, GA). B. anthracis Pasteur was cultured in a dialysate of brain heart infusion broth and 0.8% NaHCO3 for 72 h on a gyratory shaker at 37°C in 15% CO2. Cultures were killed by treatment with 4% formaldehyde for 72 h at 37°C and subsequently plated on 5% sheep blood agar to ensure nonviability. DPGA was isolated from the supernatant fluid by differential precipitation with ethanol (17, 18) and CTAB (hexadecyltrimethylammonium bromide; Sigma, St. Louis, MO). Briefly, clarified supernatant fluid was acidified by the addition of 10% (wt/vol) sodium acetate and 1% (vol/vol) acetic acid, and the DPGA was recovered by precipitation with 2 volumes of ethanol. The precipitate was rehydrated in water, and the DPGA was precipitated by the addition of 1 volume of 1% CTAB in water. The precipitate was dissolved in 1 M NaCl, and sodium acetate and acetic acid were added as described above. Finally, the high-molecular-weight fraction was precipitated by the addition of 1 volume of ethanol.
Collection of tissues, serum, and urine.
Pathogen-free, 8-week-old female BALB/c mice were used for all studies (Charles River Laboratories, Inc., Wilmington, MA). For tissue distribution and clearance experiments, DPGA (250, 50, or 10 µg) was injected intravenously in 200 µl of Dulbecco's phosphate-buffered saline (dPBS) (Mediatech, Inc., Herndon, VA). The use of laboratory animals for this purpose was approved by the University of Nevada, Reno, Institutional Animal Care and Use Committee and was compliant with relevant federal guidelines. For serum clearance experiments, mice were injected intravenously with 500, 100, or 20 µg of DPGA. Treatment doses of DPGA were chosen to replicate the range of serum concentrations of DPGA found during the course of a murine model of pulmonary anthrax (17). Mice were euthanized by CO2 narcosis. The liver, spleen, kidneys, and lungs were removed and placed in either 10% buffered formalin for the preparation of paraffin-embedded slides or in 6 ml of dPBS to be homogenized for antigen immunoassay. Blood samples were collected via cardiac puncture, and serum samples were isolated. Urine samples were collected just prior to death. The samples to be assayed for DPGA content were stored in dPBS at –20°C until analysis.
Antigen capture immunoassay.
A quantitative antigen capture enzyme-linked immunosorbent assay (ELISA) for DPGA was constructed using monoclonal antibody (MAb) F24F2 immunoglobulin G3 (17, 18). Tissues were homogenized in dPBS using an Omni TH homogenizer (Omni International, Waterbury, CT). Microtiter plates were coated overnight with 100 µl of MAb F24F2 (0.75 µg/ml), washed with 0.05% Tween 20 in PBS (PBS-Tween), and blocked for 90 min with PBS-Tween. Samples of serum, urine, or supernatant fluids from tissue homogenates were serially diluted in PBS-Tween and incubated for 90 min with the MAb-coated wells. Plates were washed with PBS-Tween, incubated for 90 min with 100 µl of horseradish peroxidase (HRPO)-labeled MAb F24F2 (0.25 µg/ml), washed, and then incubated with tetramethylbenzidine substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD). The reaction was stopped with a solution of 1 M H3PO4. The plates were read using a VersaMax plate reader (Molecular Devices, Sunnyvale, CA). Concentrations of DPGA in the samples were calculated using purified B. anthracis DPGA as a standard with the assistance of Softmax Pro (Molecular Devices). An optical density at 450 nm (OD450) of 0.5 was used as the end point. Values were adjusted to account for background. The sensitivity of the antigen capture ELISA was approximately 200 pg DPGA per ml.
Results from the analysis of tissues are reported as micrograms DPGA per tissue. All estimates of the amounts of DPGA in tissues were corrected for DPGA found in the plasma volume in each tissue (8). Estimates of serum DPGA were calculated in a manner similar to that of Grinsell et al. (10), assuming that the blood volume for a mouse is 5.77 ml/100g, that half of the blood volume is plasma, and that the average weight of each mouse is 20 g. A preliminary experiment was done in which tissues were spiked with purified DPGA. A comparison of immunoassays using purified DPGA alone and DPGA in the presence of tissue homogenates showed no effect of the tissue homogenates on assay performance.
Data analysis.
Plots of DPGA clearance were generated using SigmaPlot 10.0 (Systat Software, Inc., Point Richmond, CA). The data were analyzed using two exponential decay models. The two-parameter exponential decay equation is y = ae–bx, where a is the y intercept (amount of DPGA present at time zero) and b is the rate constant for clearance. The four-parameter exponential decay model is y = ae–bx + ce–dx, where a is the proportion of DPGA that clears rapidly during the initial clearance step, b is the rate constant for early clearance, c is the proportion of DPGA that clears more slowly, and d is the rate constant for the slower clearance step.
Data from the analysis of serum are also reported as a clearance rate per hour. The two-parameter exponential decay model was applied, and the clearance constant was used to determine the amount of DPGA that was cleared at each of the hourly time points. The amount of DPGA cleared per hour was determined by multiplying the amount of DPGA at the start of the hour by the rate constant.
Immunohistochemistry (IHC).
HRPO- and Alexa Fluor 555-labeled MAb F24F2 were used to identify the sites of DPGA binding. MAb F24F2 was coupled to Alexa Fluor 555, according to the manufacturer's instructions (Invitrogen, Carlsbad, CA), or HRPO using the EZ-Link Plus activated peroxidase kit (Pierce, Rockford, IL). Fluorescein isothiocyanate (FITC)-labeled F4/80 antibody (Cedarlane, Ontario, Canada) was used as a general marker for macrophages (1, 20). Tissues were fixed in 10% buffered formalin, paraffin embedded, and sectioned (3 µm). Tissue sections were mounted on slides, deparaffinized using a xylene/alcohol gradient, rehydrated in H2O, and blocked for 15 min with 5% (vol/vol) goat serum in PBS. Slides were then incubated for 1 h with either Alexa Fluor 555- or HRPO-labeled MAb F24F2 or antimacrophage FITC F4/80 (1). For dual staining, the slides were incubated for 1 h with each antibody. 3-Amino-9-ethylcarbazole substrate (Vector Labs, Burlingame, CA) was used to develop HRPO-stained slides, and hematoxylin was used as a counterstain. Slides were viewed with a Nikon Eclipse E800 epifluorescence microscope, and images were obtained using a Nikon C1 confocal system. Images were processed using Simple PCI 5.1 (Compix, Inc., Sewickley, PA).
Macrophage ablation.
Liposomes containing clodronate (Cl2MBP) were prepared as described previously (27). Liposomes were injected intravenously in a 200-µl volume. PBS was injected as a control. Control experiments, using India ink uptake as a marker for macrophages (12, 28), showed an absence of macrophages 48 h after treatment (not shown).
Molecular sieve chromatography.
A Superdex 200 molecular sieve (GE Healthcare, Piscataway, NJ) with a running buffer of PBS-Tween (0.05%) was used to compare the molecular sizes of native DPGA, DPGA in serum, and DPGA excreted in urine. The column was loaded with 100 ng of each DPGA sample in 500 µl of running buffer. The concentrations of DPGA in serum and urine were determined by an ELISA in comparison with a standard curve of purified DPGA. One-hour serum and urine samples were examined due to the high DPGA concentrations in those fluids at that time point. The amounts of DPGA in the fractions were determined by antigen capture ELISA; the results are reported as the OD450 for each fraction.
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RESULTS
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DPGA distribution in tissues, serum, and urine.
Mice were injected intravenously with various doses of DPGA in PBS. Samples of tissues (liver, spleen, kidney, and lung), blood, and urine were collected at various intervals after treatment, and the amount of DPGA in each sample was determined by quantitative antigen capture ELISA. The results (Fig. 1) showed the liver to be the primary tissue depot for DPGA, accounting for almost all of the injected antigen. Spleen and kidney samples showed smaller amounts of DPGA. The lung samples were also examined; negligible amounts were detected (data not shown). At the 250-µg dose, DPGA reached a maximum level in the liver by day 2, whereas the 50- and 10-µg doses peaked at 12 and 8 h, respectively. Immunoassays done using tissues from untreated mice showed values at background levels (data not shown).
DPGA was also present in urine samples, with the highest concentrations found shortly after injection, followed by a low level of residual excretion over several days. Samples of feces were also examined; no DPGA was detected (data not shown).
Plots were generated to evaluate the clearance of DPGA from the liver, spleen, kidneys, and urine as a function of time (Fig. 2). Analysis began at the time point when DPGA reached a maximum in the tissue or urine and ended when DPGA values dropped below the detection limit. For the liver, the best fit for the data was a biphasic exponential decay model (y = ae–bx + ce–dx) which suggested two distinct clearance rates. An initial half-life of 6 h and a secondary half-life of 4.6 days were found for DPGA in the liver of mice given the 250-µg dose (Table 1 and Fig. 2). For the 50-µg dose, the half-lives in the liver were 1.5 and 8.2 days. The 10-µg dose exhibited half-lives of 12 h and 3 days.
The best fit for the clearance of DPGA from the spleen was obtained using a two-parameter exponential decay equation (y = ae–bx) which suggested a single clearance rate. The half-lives for DPGA in the spleen for the 250-, 50-, and 10-µg doses were 4.1 days, 2.5 days, and 14 h, respectively (Table 1). The clearance of DPGA from the kidneys also best fit a two-parameter exponential decay model. The half-life for the 250-µg dose was 3 days; half-lives for the 50- and 10-µg doses were 18 and 9 h, respectively (Table 1).
The clearance of DPGA from the urine was calculated from 1 to 12 h postinjection (Fig. 2). After 12 h, DPGA was still found in the urine but at a relatively low level for which a clearance rate could not be determined. The half-lives of DPGA in the urine were similar for the three doses: 2 days, 1.5 days, and 2.8 days.
Clearance from the serum was very rapid, dropping below detectable levels at or before 24 h with all three doses (Fig. 3). Serum levels following the injection of 20 µg DPGA were too low to allow for the calculation of a clearance rate. Despite noticeably different half-lives for mice given 500 µg (5 h) or 100 µg (1.3 h), the rates of clearance were similar for the two treatment doses (140 versus 110 µg/h).
Sites of tissue deposition.
Tissues were harvested at days 1, 2, 4, and 8 after the intravenous injection of 500, 100, or 20 µg of DPGA, and the sites of DPGA deposition were evaluated by IHC. Immunohistochemical staining with HRPO-labeled MAb F24F2 showed the deposition of DPGA in the liver to be localized primarily to the endothelial cells of the sinusoids (Fig. 4). Deposition in the spleen was found primarily in the red pulp (Fig. 5). Dose size did not appear to affect sites of tissue localization. Clearance trends were similar for all three doses and tracked the clearance patterns shown by quantitative immunoassay (Fig. 1). Trace amounts were found in the glomeruli of the kidneys (not shown).
Studies of capsular polysaccharides have found liver Kupffer cells to be a major site for antigen accumulation (9, 10, 16). As a consequence, the potential for DPGA accumulation in the Kupffer cells was examined in more detail. In this experiment, Alexa Fluor 555-labeled MAb F24F2 was used to identify DPGA, and the general macrophage marker FITC F4/80 identified Kupffer cells. The results showed the binding of DPGA to the Kupffer cells in the liver (Fig. 6). However, the majority of cells showing an accumulation of DPGA, i.e., sinusoidal endothelial cells, were not stained with F4/80 antibody. The injection of liposomes containing clodronate to deplete Kupffer cells was done to further evaluate the accumulation of DPGA by Kupffer cells. Macrophage depletion was confirmed by the assessment of the cellular uptake of India ink; treated mice showed no uptake of India ink in the liver (not shown). Similarly, liver samples from liposome-treated mice showed no staining with FITC F4/80 antibody (Fig. 6). Macrophage-depleted livers exhibited a high level of DPGA accumulation in the sinusoids (Fig. 6). Control tissues from mice not treated with DPGA showed no staining with the DPGA-specific MAb (data not shown).
In vivo degradation of DPGA.
The excretion of DPGA in the urine (Fig. 1) suggested that the native DPGA used for treatment may be degraded in vivo. As a consequence, we used molecular sieve chromatography to compare the molecular size of the injected DPGA to the DPGA found in serum or urine. Both native DPGA and DPGA found in serum ran at or near the void volume (Fig. 7). In contrast, DPGA in urine was of a much smaller and more heterogeneous molecular size than native DPGA. In an effort to evaluate the size of the excreted DPGA, we determined the elution volume of a synthetic 25-mer peptide of DPGA. The synthetic 25-mer eluted in fractions 52 to 56. Since molecular size might influence the sensitivity of the ELISA for antigen detection, we compared the sensitivity of the ELISA for the detection of native DPGA and synthetic DPGA. The results showed an eightfold loss of sensitivity for the 25-mer. This result suggests that the amount of DPGA found in urine is likely an underestimate of the amount of excreted DPGA due to the loss of assay sensitivity with a reduction in the size of the antigen.
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DISCUSSION
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Several previous studies examined the in vivo distribution of capsular antigens (9, 10, 16, 21). In perhaps the first such study, Kaplan et al. examined the localization of pneumococcal polysaccharide types 2 and 3 in mouse tissues (16). The results showed accumulations of antigen in splenic macrophages, Kupffer cells in the liver, and inguinal lymph node macrophages. A study of the tissue localization of Haemophilus influenzae type b capsular polysaccharide found rapid deposition of antigen in the Kupffer cells of liver sinusoids and in the macrophages of the red pulp of the spleen and the sinuses of lymph nodes (13). More recently, studies done by Grinsell et al. (10), Lendvai et al. (21), and Goldman et al. (9) found similar results with glucuronoxylomannan, the major capsular polysaccharide from Cryptococcus neoformans. Our study differs from these previous reports, because we examined the fate of a capsular polypeptide rather than a polysaccharide. Similar to studies of capsular polysaccharides, our results found DPGA primarily in the liver and spleen.
While our results are similar to previous studies of capsular polysaccharides in that the liver is a main depot for DPGA accumulation, there was a substantial difference in the rates of clearance of antigen from the tissues and fluids. Capsular polysaccharides have previously been found to remain in the blood and tissues much longer than we found with our studies of DPGA. Kaplan et al. found pneumococcal polysaccharide in most murine tissues up to 75 days after injection (16). Grinsell et al. found that cryptococcal polysaccharide remained in the serum for days after injection and did not completely clear from the liver, spleen, and kidneys even after 1 month postinjection (10). In contrast, our studies of DPGA showed rapid serum clearance, with a half-life of about 5 h for the high dose, and complete tissue clearance by 21 days. This rapid clearance from the blood and tissues suggests that the presence of DPGA in serum may indicate an active source of antigen production in the body.
The half-life for the clearance of DPGA from serum depended on the treatment dose; high doses produced more-extended half-lives (Fig. 3). In contrast, the actual clearance rate per hour was largely independent of the treatment dose (Fig. 3). We interpret these results to indicate that there is a fixed rate for the clearance of DPGA. When the amount of DPGA in serum exceeds this potential clearance rate, DPGA accumulates faster than it can be cleared. As a consequence, the high level of DPGA produced during an active B. anthracis infection will likely result in very high serum levels of capsular antigen. This is the result that we observed in a murine model of pulmonary anthrax where serum DPGA levels approached 1 mg/ml serum (17). We have observed similar high levels of serum DPGA in rabbit and nonhuman primate models of pulmonary anthrax (unpublished results).
We used IHC to identify sites of DPGA accumulation in the liver. Our results showed that the liver had a much higher concentration of DPGA than the spleen, and nearly undetectable amounts were found in the kidneys. We found that Kupffer cells and sinusoidal endothelial cells accumulate DPGA in the liver. DPGA also accumulated in the red pulp in the spleen. Macrophage ablation studies verified that, in the absence of macrophages, the liver remains a major depot for DPGA, thereby identifying multiple cellular sites for DPGA binding in the liver. Finally, it should be noted that we cannot exclude the possibility that DPGA is taken up by other cell types, e.g., dendritic cells that might be present in lower numbers or that might take up lesser amounts of DPGA.
Previous studies have also shown that the hepatic sinusoidal endothelial cells (HSEC) can contribute to scavenger and nonspecific immune functions, cooperating with the Kupffer cells to eliminate certain macromolecules (6, 26). Indeed, in combination with Kupffer cells, HSEC constitute the most powerful scavenger system in the body (23). The uptake of specific classes of solutes is facilitated by the expression of multiple scavenger receptors (19). For example, HSEC are the sites for clearance of hyaluronic acid from the bloodstream (7). Hyaluronic acid has several similarities to DPGA, including (i) the presence of a repeating structure (glucuronic acid and N-acetylglucosamine), (ii) a negative charge, and (iii) a high molecular weight. It is possible that the lack of accumulation of capsular polysaccharides by HSEC may account for the more rapid clearance of DPGA from the circulation compared with the slower clearance of capsular polysaccharides.
We found that DPGA was excreted in the urine in a degraded form and was detectable in the urine for several days after injection. No detectable DPGA was found in the feces. These results are consistent with studies done by Coonrod (3) and Dochez and Avery (4), who found that pneumococcal capsular polysaccharide was also excreted in the urine at high concentrations. Coonrod noted that polysaccharide excretion in the urine can persist for days to weeks due to the slow breakdown of polysaccharide in the tissues. Given the fact that a 25-mer of DPGA displayed reduced reactivity in the antigen capture ELISA, it is likely that the calculated levels of DPGA in urine are underestimates of the actual amount of DPGA excreted in urine.
Finally, our results have several implications for the detection of DPGA for the immunodiagnosis of anthrax. In a murine model of pulmonary anthrax, DPGA was found in the serum at concentrations approaching 1 mg/ml (17). Since DPGA is shed into the blood at such high concentrations, detection of DPGA in the serum has the potential to be a diagnostic marker for an active B. anthracis infection. Rapid clearance of DPGA from serum suggests that the presence of DPGA in serum reflects an active source of DPGA synthesis, i.e., a B. anthracis infection. Moreover, the apparent degradation of DPGA and its excretion in urine raise the possibility that urine could be used as a noninvasive sample for rapid screening for a B. anthracis infection. As a consequence, the detection of DPGA in these fluids may provide a rapid means for diagnosis, leading to reduced morbidity and mortality associated with human cases of anthrax.
In summary, the in vivo clearance of DPGA has several similarities to the clearance of capsular polysaccharides; the liver and spleen are the primary clearance organs, and both accumulate in tissue macrophages. There are also important differences: DPGA appears to accumulate in HSEC, whereas previous studies of capsular polysaccharides showed no apparent accumulation in HSEC. In addition, DPGA shows a much more rapid clearance from the circulation than that which was found in clearance studies of capsular polysaccharides.
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ACKNOWLEDGMENTS
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This study was supported by Public Health Service grants R01-AI059348 and U01-AI061200 from the National Institute of Allergy and Infectious Diseases. We thank the Nevada State Health Laboratory for assistance in preparing our IHC slides.
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FOOTNOTES
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* Corresponding author. Mailing address: Department of Microbiology and Immunology/320, University of Nevada School of Medicine, Reno, NV 89557. Phone: (775) 784-4124. Fax: (775) 327-2332. E-mail: tkozel{at}medicine.nevada.edu 
Published ahead of print on 14 January 2008.
Editor: A. Casadevall
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Infection and Immunity, March 2008, p. 899-906, Vol. 76, No. 3
0019-9567/08/$08.00+0 doi:10.1128/IAI.01176-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
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ABSTRACT
INTRODUCTION
