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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.
-D-Glutamic Acid, the Capsular Antigen from Bacillus anthracis
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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-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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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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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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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).
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FIG. 1. DPGA accumulation and distribution in tissues over time. Mice were injected intravenously with 250, 50, or 10 µg of DPGA (PGA). Liver, spleen, kidney, and urine samples were collected at the indicated time points and assayed by a quantitative ELISA for DPGA. Data shown are the mean ± standard deviation for five mice per time point. Data are representative of three independent experiments with similar results.
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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.
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FIG. 2. Kinetics for clearance of DPGA from tissues and urine. Clearance plots begin with the time point at which DPGA (PGA) accumulation reached a maximum in each tissue and urine (as shown in Fig. 1). Curves were generated using exponential decay models. Clearance from the liver was best described by a four-parameter exponential decay model, whereas a two-parameter exponential decay model showed a best fit for spleen, kidneys, and urine. n = 5 mice per time point for each concentration. Data are representative of three independent experiments with similar results.
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TABLE 1. Tissue half-lives following intravenous injection of DPGA
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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).
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FIG. 3. Kinetics for clearance of DPGA (PGA) from serum. Mice were injected intravenously with 500, 100, or 20 µg of DPGA. Animals were euthanized at the indicated time points, and blood samples were collected via cardiac puncture. Clearance rates per hour were calculated using the rate constant from an exponential decay model. n = 5 mice per time point for each concentration. Data are representative of five independent experiments with similar results. t1/2, half-life.
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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).
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FIG. 4. IHC showing deposition of DPGA in the liver. Mice were injected intravenously with 500, 100, or 20 µg of DPGA. Tissues were harvested at days 1, 2, 4, and 8. DPGA localization was identified by use of HRPO-labeled MAb F24F2 (brown). Hematoxylin was used as a counterstain. Control shows staining of tissue from mice that were not treated with DPGA. Note the deposition of DPGA in the sinusoids that surround the liver hepatocytes.
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FIG. 5. IHC showing deposition of DPGA in the spleen. Mice were injected intravenously with 500, 100, or 20 µg of DPGA. Tissues were harvested at days 1, 2, 4, and 8. DPGA localization was identified by use of HRPO-labeled MAb F24F2 (brown). Hematoxylin was used as a counterstain. The control shows the staining of tissue from mice that were not treated with DPGA. Treatment with 20 µg DPGA did not provide sufficient staining for the localization of antigen. Note the deposition of DPGA in the red pulp.
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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).
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FIG. 6. Cellular location of DPGA in the liver. Mice were injected intravenously with 50 µg of DPGA (top panels), and livers were harvested after 24 h. DPGA is identified with Alexa Fluor 555-labeled MAb F24F2 (left panels). Anti-F4/80 FITC was used to label the Kupffer cells (center panels). A merged image (top right panel) shows two Kupffer cells that have taken up DPGA (arrows) as well as numerous additional cells that have also accumulated DPGA. Mice were treated intravenously with liposome encapsulated clodronate (bottom panels) to deplete the Kupffer cells. After 48 h, mice were injected intravenously with 50 µg of DPGA, and tissues were collected 24 h later and stained for DPGA and Kupffer cells as described above. Note the binding of DPGA in the absence of Kupffer cells.
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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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FIG. 7. Relative molecular size of native DPGA (PGA) isolated from B. anthracis or DPGA in serum and urine 1 h after injection of 100 µg native DPGA. Samples from three animals were pooled for analysis. The samples were normalized such that 100 ng of DPGA was loaded onto a Superdex 200 column. The relative amount of DPGA in each fraction is represented by the OD450 when a sample (100 µl) of each fraction was analyzed by an antigen capture ELISA. Vo is the fraction at which the void volume elutes from the column.
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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.
Published ahead of print on 14 January 2008. ![]()
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-D-glutamic acid capsule covalently coupled to a protein carrier using a novel triazine-based conjugation strategy. J. Biol. Chem. 281:4831-4843.This article has been cited by other articles:
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