An original, reliable, and reproducible method for the purification of Enterocytozoon bieneusi spores from human stools is described. We recently reported the ...
JOURNAL OF CLINICAL MICROBIOLOGY, May 2001, p. 1947–1951 0095-1137/01/$04.00⫹0 DOI: 10.1128/JCM.39.5.1947–1951.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 39, No. 5
One-Step Purification of Enterocytozoon bieneusi Spores from Human Stools by Immunoaffinity Expanded-Bed Adsorption ISABELLE ACCOCEBERRY,1* MARC THELLIER,2 ANNICK DATRY,2 ISABELLE DESPORTES-LIVAGE,2 SYLVESTRE BILIGUI,2 MARTIN DANIS,2 AND XAVIER SANTARELLI3 Laboratoire de Parasitologie-Mycologie, Centre Hospitalier-Universitaire de Bordeaux, Ho ˆpital Saint Andre´, 33075 Bordeaux Cedex,1 Unite´ INSERM 511 and Laboratoire de Parasitologie-Mycologie, Centre Hospitalier-Universitaire de la Pitie´-Salpeˆtrie`re, 75013 Paris,2 and Ecole Supe´rieure de Technologie des Biomole´cules de Bordeaux (ESTBB) UMR 5544, Universite´ Victor Segalen de Bordeaux 2, 33076 Bordeaux Cedex,3 France Received 12 October 2000/Returned for modification 14 January 2001/Accepted 26 February 2001
An original, reliable, and reproducible method for the purification of Enterocytozoon bieneusi spores from human stools is described. We recently reported the production of a species-specific monoclonal antibody (MAb) 6E52D9 immunoglobulin G2a (IgG2a) raised against the exospore of E. bieneusi spore walls. The MAb was used as a ligand to develop an immunoaffinity matrix. The mouse IgG2a MAb was bound directly to a Streamline rProtein A adsorbent, used for expanded-bed adsorption of immunoglobulins, for optimal spatial orientation of the antibody and maximum binding efficiency of the antigen. The complex was then cross-linked covalently using dimethyl pimelimidate dihydrochloride. After incubation of the immunoaffinity matrix with filtered stool samples containing numerous E. bieneusi spores and before elution with 6 M guanidine HCl, the expansion of the adsorbent bed eliminated all the fecal contaminants. The presence of spores in the elution fractions was determined by an indirect immunofluorescence antibody test (IFAT). E. bieneusi spores were found in the elution fraction in all four experiments and were still highly antigenic as indicated by IFAT. Smears examined by light microscopy contained very clean spores with no fecal debris or background bacterial and fungal contaminants. However, spore recovery rates were relatively low: an average of 107 spores were purified per run. This technique for isolating E. bieneusi spores directly from human stool samples with a high degree of purity opens up new approaches for studying this parasite. Microsporidia are obligate intracellular protistan parasites that infect a variety of cells in a wide range of invertebrate and vertebrate hosts. Over 1,000 species have been described but, until recently, reports of human infections were rare. Since the AIDS epidemic, microsporidia have been identified as major opportunistic pathogens. In 1985, the microsporidian species most commonly found in humans was identified as Enterocytozoon bieneusi (11). This parasite is usually observed in the human immunodeficiency virus (HIV)-infected patients with CD4 lymphocyte counts of less than 50 cells per mm3, complaining of chronic diarrhea, nausea, malabsorption, and severe weight loss (4, 28). Encephalitozoon intestinalis (19) also causes intestinal infections frequently associated with nephritis, sinusitis, or bronchitis (23, 24). Significantly, the introduction of highly active antiretroviral therapy (HAART) is able to greatly modify the course of intestinal microsporidiosis in patients infected with HIV (15, 25; J. Goguel, C. Katlama, C. Sarfati, C. Maslo, C. Leport, and J. M. Molina, Letter, AIDS 11:1603–1610, 1997). However, even in the HAART era, diarrhea is still a debilitating symptom in HIVinfected patients (3). These parasites are also pathogenic in subjects with immunodeficiency due to other causes than AIDS. Cases of intestinal microsporidiosis have been detected in organ transplant recipients (30, 34). The two species E. bieneusi and E. intestinalis also appear to be responsible for cases of diarrhea in
immunocompetent subjects (16). Most of these are travelers returning from tropical areas (31, 33, 37, 43). More surprisingly, an increasing number of HIV-seronegative and asymptomatic individuals have been found to be infected with microsporidia (13, 20, 42). Immunological tools remain helpful for diagnosis, epidemiological survey, and experimental investigation. E. intestinalis isolates are easily obtained through in vitro systems (40) but, to date, such systems are still lacking for E. bieneusi. In the absence of an in vitro cultivation model and in view of the invasive procedures needed for collecting epithelium or fluid samples from the gastrointestinal tract, human stools are the most widely used source of E. bieneusi spores for diagnosis and research. We have recently reported the production of a species-specific monoclonal antibody (MAb) 6E52D9 immunoglobulin G2a (IgG2a) raised against the exospore of E. bieneusi spore walls. This MAb detects this agent in human stool specimens (1). In this report, we describe the development of an immunoaffinity matrix, using the IgG2a MAb and a recombinant protein A adsorbent, designed for expanded-bed adsorption (EBA) technology (10, 12), to purify E. bieneusi spores directly from human stools. All fecal debris and bacterial and fungal contaminants are eliminated in the flow-through when the matrix is expanded, and the E. bieneusi spores can be eluted with a high degree of purity.
* Corresponding author. Mailing address: Laboratoire de Parasitologie-Mycologie, CHU de Bordeaux, 33000 Bordeaux, France. Phone: 33-5-56-79-58-37. Fax: 33-5-56-79-58-79. E-mail: isabelle.accoceberry @chu-bordeaux.fr.
E. bieneusi sources. Fecal specimens were obtained from HIV-infected patients. Microsporidian spores were detected by fluorochrome Uvitex 2B stain (41) and Weber’s chromotrope-based modified trichrome stain (22). Fecal sam-
MATERIALS AND METHODS
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ples containing numerous small oval spores were homogenized and suspended in a solution of phosphate-buffered saline (PBS; Sigma Laboratories, Saint-Quentin-Fallavier, France). The samples were processed for transmission electron microscopy (TEM) and tested by PCR to confirm the identification of the species and ensure that there was no concomitant E. intestinalis infection. TEM. The fecal samples were fixed at room temperature in 2.5% glutaraldehyde in 0.1 M Na cacodylate buffer (pH 7.2) for 60 min, rinsed in buffer, and then postfixed in ferriosmium [1% (wt/vol) OsO4 and K3Fe(Cn)6 in cacodylate buffer] for 60 min. After ethanolic dehydration, the samples were embedded in Spurr resin. Thin sections, stained with uranyl acetate and lead citrate, were examined with a JEOL JEM 100 CX transmission electron microscope. PCR amplification. The PCR assay was performed as described previously by Ombrouck et al. (27). V1 (5⬘-CACCAGGTTGATTCTGCCTGAC-3⬘) and EB450 (5⬘-ACTCAGGTGTTATACTCACGTC-3⬘) primers, described by Zhu et al. (45), were used to amplify E. bieneusi DNA. V1 and SI500 (5⬘-CTCGCT CCTTTACACTCGAA-3⬘) primers, described by Weiss et al. (44), were used to amplify E. intestinalis DNA. Processing spore samples. The stool suspensions were filtered through a graded series of five nylon sieves (pores size of 100, 50, 30, 20, and 10 m). Filtration was facilitated by adding 1,000 to 2,000 ml of PBS. The final filtrate was centrifuged at 500 ⫻ g for 6 min to eliminate large particles, and the sieved spores in the supernatant were pelleted by centrifugation at 2,500 ⫻ g for 20 min. The pellet was resuspended in PBS (1/3 [vol/vol]). Penicillin (5 IU/ml) and streptomycin (100 g/ml) were added to the final stool suspensions. Spores were counted in 2-l droplets applied to 5-mm wells on multiwell slides, using the indirect immunofluorescence antibody test (IFAT) as described below. MAbs. Two species-specific MAbs of E. bieneusi spore walls were produced as before (1). 6E52D9, isotyped as IgG2a, was directed against the exospore, and 3B82H2, isotyped as IgM, was directed against the endospore. The MAbs were expanded in culture as previously described (1) and purified from hybridoma culture supernatants by affinity protein A chromatography (39) for the 6E52D9 MAb and with Dynabead M-450 rat anti-mouse IgM (Dynal, Compie`gne, France), according to the manufacturer’s instructions, for the 3B82H2 MAb. The purified supernatants were stored at ⫺20°C until their use in the IFAT. The 6E52D9 IgG2a was used as ligand in the immunoaffinity process. A total of 2 ⫻ 106 cells from the hybridoma line were injected via the intraperitoneal route into pristane-primed female BALB/c mice (Charles River Laboratories, Saint-Aubain-les-Elbeuf, France) to produce ascitic fluid that was collected 10 to 15 days later (18). The ascitic fluids generated were incubated 1 h at 37°C and overnight at 4°C and then centrifuged at 3,000 ⫻ g for 10 min (18). The supernatants were carefully collected and screened by IFAT using purified E. bieneusi spores, as previously described (1). Ascitic fluids yielding titers greater than 1,024 were pooled (73 ml), precipitated by adding an equal volume of saturated ammonium sulfate (18, 36), and incubated at 4°C for 4 h. The purified mouse immunoglobulin was recovered by centrifugation at 10,000 ⫻ g at 4°C for 20 min. The pellet was dissolved in a small volume of 0.05 M Tris-HCl (pH 9) and injected into a desalting Sephadex G-25 column (Amersham Pharmacia Biotech, Saclay, France) equilibrated with 1 M NaCl–0.05 M Tris-HCl (pH 9) to remove the residual ammonium sulfate and condition the MAb in the binding buffer (29). Immunoglobulin content was determined by absorbance at 280 nm using a UV spectrophotometer. Chromatographic system and EBA method. The chromatographies were performed with fast-protein liquid chromatography and Biopilot workstations (Amersham Pharmacia Biotech). The Streamline rProtein A matrix (Amersham Pharmacia Biotech) is used for EBA of immunoglobulins (38). rProtein A is a recombinant protein. The base matrix is a 4% agarose derivative with an inert metal alloy core that provides the density required to use the adsorbent in expanded-bed mode. These porous beads have a size distribution of 80 to 165 m and a particle density of 1.3 g/ml. The matrix was poured into a Streamline 25 column (Amersham Pharmacia Biotech) designed for the EBA technology (9, 10, 12). This is a glass column with an inner diameter of 25 mm, with a specially designed liquid distributor at the base of the column and a top mobile adapter. The bed is expanded by upward liquid flow. Adsorbent particles are suspended in equilibrium due to the balance between particle sedimentation velocity and upward flow. The sample is applied to the expanded bed with an upward flow. Target molecules are captured on the adsorbent while cell debris, cells, particulates, and contaminants pass through unhindered. Flow is then reversed. The adsorbent particles settle quickly and target molecules are desorbed by an elution buffer, as in conventional packed-bed chromatography.
J. CLIN. MICROBIOL. Preparation of antibody-protein A matrix. The matrix was prepared as previously described (32, 35). A total of 75 ml of Streamline rProtein A was packed in a 2.6-by-14-cm XK 26/20 column (Amersham Pharmacia Biotech). A total of 75 ml of the MAb solution (4.5 mg/ml) in 1 M NaCl–0.05 M Tris-HCl (pH 9) binding buffer was recirculated through the column at a flow rate of 0.5 ml/min for 4 h at room temperature. The column was washed at the same flow rate with 250 ml of PBS and with 250 ml of 0.2 M triethanolamine HCl buffer at pH 8.5. A total of 100 ml of 0.2 M triethanolamine HCl buffer (pH 8.5) containing 520 mg of dimethyl pimelimidate dihydrochloride (Sigma Laboratories) was recycled through the column at 0.5 ml/min for 4 h. The column was then washed at the same flow rate with 200 ml of PBS, 200 ml of 0.2 M triethanolamine (pH 8.5), 200 ml of 4 M guanidine HCl and, finally, with 500 ml of PBS. The presence of the MAb was checked in all of the washing fractions by IFAT using purified E. bieneusi spores, as previously described (1). Purification of E. bieneusi spores using EBA technology. EBA technology was performed in the Streamline 25 columnn containing 75 ml of the synthesized immunoaffinity matrix, using a Biopilot workstation. Spore suspension (75 ml) was injected into the column and incubated with the gel at room temperature overnight. The gel was expanded and washed, to remove all fecal particles and unbound spores, at an upward flow velocity of 32 ml/min, until the UV baseline was reached. PBS buffer (pH 7.2) was used during expansion and washing. The workstation pump was then turned off and the bed sedimented. The column adapter was moved down toward the sedimented bed surface. After a wash with PBS, the run was performed at a downward flow rate of 15 ml/min. The elution buffer was run at the same flow rate. Several potential elution buffers were tested to determine the proper conditions (18): glycine at 50 mM (pH 2.49), ethylene glycol at 25%, 4 M guanidine HCl, and 6 M guanidine HCl. The elution fractions were then collected into 50-ml Falcon centrifuge tubes, sedimented at 2,500 ⫻ g for 20 min, and washed four times by centrifugation in PBS to remove residual elution buffer. The pellets were pooled, resuspended in 5 ml of PBS, and stored at 4°C until used. The column was reequilibrated to the starting buffer by passing 20 column volumes through the matrix and 0.02% sodium azide was added for long-term storage at 4°C. Characterization of the concentrated elution fraction. (i) IFAT. The concentrated elution fraction was used as antigen. IFAT was performed using purified hybridoma culture supernatants of the two MAbs specific to E. bieneusi spore walls. Briefly, the elution fraction was applied to 18-well slides (2 l per 5-mm well) and incubated sequentially with purified supernatants, diluted at 1:64 in 0.1% bovine serum albumin in PBS, and fluorescein isothiocyanate-labeled goat antimouse IgG-IgM-IgA (1:200 dilution; Sigma Laboratories). Slides were washed, mounted with buffered glycerol mounting fluid, and examined with a Leitz Laborlux fluorescence epifluorescence microscope (1). (ii) Light microscopy. In addition, two smears were prepared and stained by fluorochrome Uvitex 2B stain (41) and Weber’s chromotrope-based modified trichrome stain (22). (iii) Testing for bacterial and fungal contamination. A sample of the concentrated elution fraction was checked for bacterial and fungal contaminants after 48 and 72 h of aerobic and anaerobic culture. (iv) Spore counting. Purified spores were counted using both a hemocytometer and IFAT.
RESULTS E. bieneusi sources. Four stool samples containing numerous small oval spores of E. bieneusi, as indicated by bright fluorescence with Uvitex 2B and proved by TEM and PCR, were selected from three HIV-infected patients and separately processed in four EBA experiments. Initial spore levels were determined by averaging several direct counts on the filtered fecal samples (Table 1). Two fecal samples were freshly collected (samples 2 and 3), while the other two had been stored in PBS at 4°C for 6 months (samples 1 and 4). MAb. A total of 73 ml of ascitic fluid was obtained. After defibrination, 337.5 mg of the MAb was recovered in 75 ml of binding buffer by precipitation with saturated ammonium sulfate and desalting chromatography.
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FIG. 1. Chromatogram of spore purification using the immuno-Streamline rProtein A matrix. Features: column, Streamline 25 (75 ml of gel); sample, 75 ml of the spore suspension; buffer, PBS at pH 7.2; detection at 280 nm (y axis); elution volume (x axis); flow rate, 2 ml/min in equilibration and injection (I), 32 ml/min in expansion (Ex), and 15 ml/min in sedimented bed for elution (El). The fractions between 500 and 1,500 ml of elution volume represent fecal debris eliminated after expansion. The peak at ca. 1,600 ml of elution volume represents the eluted spores.
Preparation of antibody-protein A matrix. The antigen-antibody complex formed between the Streamline rProtein A and the IgG2a raised against E. bieneusi spores was easily crosslinked with dimethyl pimelimidate dihydrochloride. After injection of the MAb into the Streamline rProtein A, no MAb was found in the flow-through. Coupling with dimethyl pimelimidate dihydrochloride was verified by washing several times with triethanolamine and guanidine. No MAb was found in any of the washing fractions. Purification using EBA technology. Spore suspension (75 ml) was injected into the sedimented bed, and the pump was stopped. The suspension was incubated with the immunomatrix overnight. The pump was turned on, and the gel was gradually expanded to 2.5 times its sedimented height by an upward flow. All of the fecal contaminants entrapped between the beads were thus eliminated by the buffer flow (Fig. 1). When the UV baseline was reached, the pump was turned off and the bed sedimented. The elution procedure relied on breaking the bonds between the spores and the MAb. Only 6 M guanidine HCl, used to remove strongly bound protein, yielded a sharp peak with complete release of the spores (Fig. 1). Two fractions, averaging 50 ml each, were recovered and then washed, concentrated, and analyzed by IFAT. Characterization of the concentrated elution fraction. (i) IFAT. The presence of spores in the elution fraction was determined by IFAT. The two MAbs showed strong indirect immunofluorescence after incubation with the final concentrated elution fraction used as antigen. Indeed, the MAbs reacted with small oval spores present in the sample which fluoresced brightly (4⫹ on a scale of 0 to 4), with prominent labeling of the spore walls (Fig. 2). E. bieneusi spores were found in the elution fraction of all four experiments. However,
relatively few spores were observed at three to five per microscopic field (⫻1,000 magnification). (ii) Light microscopy. The spores seemed highly purified. Smears examined by light microscopy contained very clean spores with no fecal debris or background bacterial and fungal contaminants. (iii) Testing for bacterial and fungal contamination. Tests for enteropathogenic bacteria and yeast cells were all negative. (iv) Spore counts. The high degree of purification of the spores made it possible to count the spores in the elution fractions using a hemocytometer, to estimate the recovery rate (Table 1) and the matrix binding capacity. The results were confirmed by IFAT. DISCUSSION Immunoaffinity purification is a powerful technique for the purification of proteins (2, 5, 8, 18, 26). However, in all cases, the samples must be clarified in some way before chromatography. Furthermore, some recently developed techniques are effective in the presence of suspended solids (6, 7). The use of EBA has the potential for efficient purification of bioproducts from unclarified feedstocks (17). EBA is a single-pass operation in which the desired molecules are purified from crude, particulate-containing feed stock with no need for prior clarification, concentration, or prepurification. The expansion of the adsorbent bed creates a distance between the adsorbent particles and increased voidage in the bed, which allows unhindered passage of cells, cell debris, and other particulates during the application of crude feed to the column. A stable expanded bed is achieved through the unique design of both the adsorbent particles and the liquid distributor at the base of the column. The size of the bead matrix and end piece net
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TABLE 1. Summary of E. bieneusi spore recovery efficiencies Sample no.
Processing sample vol (ml)
No. of spores/ sample
No. of spores recovered/eluted fractiona
% E. bieneusi spores recovered
1 2 3 4
30 28 31 32
5.1 ⫻ 108 9.8 ⫻ 108 6.2 ⫻ 108 8.3 ⫻ 108
8.75 ⫻ 106 1 ⫻ 107 1 ⫻ 107 1.25 ⫻ 107
1.71 1.02 1.61 1.5
a In each experiment, the eluted fractions were concentrated and resuspended in a volume of 5 ml of PBS.
allowed cells or other contaminants to pass through. Since human stools are the source of E. bieneusi, spores are always contaminated with fecal debris, bacteria, and yeast cells, irrespective of the recovery technique. We recently produced a mouse IgG2a MAb (1) raised against the exospore of E. bieneusi spore walls, so we decided to create an immunoaffinity matrix using this MAb as a ligand for EBA purification of the E. bieneusi spores. To optimize the interaction between the MAb and the spores, the Fab fragments of the immunoglobulin must be free; therefore, the MAb must be oriented in a correct way, linked to the matrix by the Fc fragment (18). The high specificity of protein A for the Fc fragment of mouse IgG2a (14) made it possible to construct a suitable immunoaffinity matrix, giving excellent one-step purification. To prevent coelution of the MAb, the link between the MAb and the protein A was reinforced by chemical coupling with a bifunctional coupling reagent, dimethyl pimelimidate dihydrochloride, which is cheap and easy to handle (32, 35). Spores must bind to MAb on a solid-phase matrix. As the antibody is not in solution, the time required for the antibodybead–antigen interaction will have different kinetics than soluble interaction (18). After several experiments, we noticed that the binding time had to be increased to between 6 and 12 h to collect the spores efficiently. We therefore modified the standard EBA procedure. A volume of spore suspension equivalent to that of the antibody matrix was injected. The flow was stopped for several hours to keep the target spores in contact with the matrix, and then expansion was performed to eliminate all of the fecal contaminants. The EBA procedure was repeated four times with similar results. An average of 107 spores were purified from freshly collected or stored stool samples. Although 6 M guanidine HCl elution buffer, a chaotropic agent, might be expected to denature the spores, they did not demonstrate any reduction in fluorescence when used as an antigen for an IFAT. The spore walls were stained bright apple green when they reacted with the two MAbs (6E52D9 labeled the exospore and 3B82H2 the endospore), indicating that these structures were still highly antigenic. The 107 spores in the elution fraction represented the binding capacity of the immunoaffinity matrix. The MAb probably bound only whole, mature spores, as demonstrated above. The binding capacity of the matrix was also reduced by different causes. Heavy spore loads in the stool samples used as feedstock contributed to reduce the accessibility of the spores to the MAb. As a result of the specific structure of the matrix
FIG. 2. Purified whole spores of E. bieneusi in the concentrated eluted fraction of sample 2, stained by indirect immunofluorescence with MAbs 6E52D9 (A) and 3B82H2 (B). Both MAbs recognize antigens localized in the spore walls. Smears were shown to contain very clean spores with no fecal debris or background bacterial and fungal contaminants (⫻1,000 magnification).
beads for the EBA technology, some IgG2a was bound to the inner matrix surface of the porous beads, probably further reducing the accessibility of the spores. In addition, 4.5 mg of the MAb was bound per ml of gel, representing only half of the saturated binding capacity of Streamline rProtein A described by the manufacturer (10 mg/ml for mouse IgG2a). The saturation of the Streamline rProtein A with the MAb at 10 mg per ml of gel and the best preparation of the initial fecal samples by using a discontinuous gradient of Percoll after the filtration procedure (1) could probably improve the interaction between the MAb and the spores. Therefore, the number of purified spores recovered would be increased. Nevertheless, this technique purifies sufficient amounts of whole spores with a high degree of purity, as large quantities of stool samples can be processed, and additionally the immunoaffinity matrix is reusable without loss of the IgG2a binding capacity, as shown by the results obtained. Further repeated use and regeneration experiments are required to determine the life expectancy and reusability of the immunomatrix (21). This study describes an efficient, reliable method for purifying E. bieneusi spores from human stool samples. The ability to obtain a pure population of whole mature spores, free of all fecal contaminants, should facilitate biological, biochemical, and immunological studies of the infective stage of E. bieneusi. Furthermore, this method is easy to scale up and could be used to process large volumes of samples (human stools, water, etc.), thus opening up new approaches for the study of E. bieneusi.
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VOL. 39, 2001 ACKNOWLEDGMENTS
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