INTRODUCTION
Microsporidia are eukaryotic, intracellular pathogens that appear to be basal fungi (James et al. Reference James, Kauff, Schoch, Matheny, Hofstetter, Cox, Celio, Gueidan, Fraker, Miadlikowska, Lumbsch, Rauhut, Reeb, Arnold, Amtoft, Stajich, Hosaka, Sung, Johnson, O'Rourke, Crocket, Binder, Curtis, Slot, Wang, Wilson, Schußler, Longcore, O'Donnell, Mozley-Standridge, Porter, Letcher, Powell, Taylor, White, Griffith, Davies, Humber, Morton, Sugiyama, Rossman, Rogers, Pfister, Hewitt, Hansen, Hambleton, Shoemaker, Kohlmeyer, Volkmann-Kohlmeyer, Spotts, Serdani, Crous, Hughes, Matsuura, Langer30, Langer, Untereiner, Lucking, Budel, Geiser, Aptroot, Diederich, Schmitt, Schultz, Yahr, Hibbett, Lutzoni, McLaughlin, Spatafora and Vilgalys2006). They are known to infect a wide range of vertebrate and invertebrate hosts with a large proportion of described species from lepidopteran and dipteran insects. Many diplokaryotic entomopathogenic microsporidia were originally assigned to the genus Nosema; more than 150 species are reported in 12 orders of insects (Becnel and Andreadis, Reference Becnel, Andreadis, Wittner and Weiss1999). Nosema bombycis, the type species of this genus (Sprague and Becnel, Reference Sprague and Becnel1998), and the first microsporidium described (Naegli, Reference Naegli1857), caused heavy losses in sericulture in Europe, America and Asia during the mid 19th century (Steinhaus and Hughes, Reference Steinhaus and Hughes1949).
Microsporidia have no vegetative stages outside their host and can reproduce only inside the host cell cytoplasm. Several stimuli, which vary among species, are known to activate the infective spores when they are ingested by the host, resulting in immediate breakdown of sporoplasm compartmentalization and germination by extrusion of the polar filament and, thus, initiation of a new infection (Undeen and Avery, Reference Undeen and Avery1984; Undeen, Reference Undeen1990). Increased hydrostatic pressure has long been thought to be the immediate cause for germination (Undeen and Vander Meer, Reference Undeen and Vander Meer1999), however, there is no information on how stimuli activate the spores.
Enriquez et al. (Reference Enriquez, Wagner, Fragoso and Ditrich1998) evaluated the effects of an anti-microsporidial exospore monoclonal antibody (mAb), 3B6, which recognizes 3 Encephalitozoon species, Encephalitozoon intestinalis, Encephalitozoon cuniculi, and Encephalitzoon hellem, on the reproduction of E. cuniculi in vitro. Pre-treatment of spores for 24 h with mAb 3B6 resulted in a 21–29% decrease in infected host cells 4 days post-infection, compared to the controls, but the protective role of antibodies in the control of microsporidial infection in this in vitro assay is not clear. Sak et al. (Reference Sak, Saková and Ditrich2004) developed a monoclonal antibody P5/H1 that recognized E. cuniculi. The effect of the P5/H1 mAb on microsporidial reproduction in a Vero E6 cell-line was a reduction in the number of E. cuniculi spores. Moreover, the presence of P5/H1 mAb increased the number of spores phagocytosed in macrophages, as measured by nitric oxide production. Thus, the surface proteins of microsporidia might influence the germination and invasion of spores. In this study, we developed mAbs to identify surface proteins that influence germination and invasion, and which might have utility for diagnostic purposes and new therapeutic strategies for controlling the microsporidian infection.
MATERIALS AND METHODS
Microsporidia
Nosema bombycis was originally isolated from infected silkworms in Zhejiang, China (Mei and Jin, Reference Mei and Jin1989). Spores were propagated in laboratory-reared silkworm larvae at 25°C and purified from the infected adults by centrifugation over Percoll as described by Canning et al. (Reference Canning, Curry, Cheney, Lafranchi-Tristem and Haque1999). Purified spores were stored in deionized water supplemented with antibiotics (Sigma, 100 U/ml penicillin, 100 μg/ml streptomycin, 2·5 μg/ml amphotericin) at 4°C and washed in phosphate-buffered saline (PBS) before use.
Monoclonal antibody production
For cell fusion, adult 7 to 9-week-old female BALB/c mice procured from Shanghai Laboratory Animal Center were intraperitoneally immunized 3 times at 14-day intervals with 108N. bombycis spores. The fusion of splenocytes and myeloma cells SP2/0 was performed using polyethylene glycol. Fused cells were maintained in RPMI-1640 containing 20% heat inactivated fetal bovine serum (FCS) (GibcoBRL Life Technologies, Grand Island, NY) and supplemented with hypoxanthine-aminopterin-thymidine (HAT) (Enriquez et al. Reference Enriquez, Bradley-Dunlop and Joens1991). Supernatants of cultures containing growing colonies were tested for the presence of antibody recognition of the specific antigen by enzyme-linked immunosorbent assay. Positive hybridomas were cloned 3 times by limiting dilution (a maximum of 5 cells per well). The clones that showed high antibody activity were injected into BALB/c mice after pristane priming for ascites production. Ascites samples were then stored in Eppendorf tubes at −20°C for further use.
The mAbs were purified from the ascites by precipitation with ammonium sulphate and dialysis against PBS (24 h at 4°C). The concentrations of the monoclonal antibodies were determined by the Bradford method (1976) using bovine serum albumin as a standard.
Enzyme-linked immunosorbent assay
The enzyme-linked immunosorbent assay (ELISA) test was performed according to the method of Hollister and Canning (Reference Hollister and Canning1987). Briefly, N. bombycis spores were incubated with appropriate hybridoma supernatants or ascites, followed by peroxidase-labelled goat anti-mouse IgG (Sigma). The reaction was developed with 0·1 m acetate substrate solution (pH 5·5) supplemented with 25 mm o-phenylendiamine (OPD) and 0·03% H2O2. The reaction was stopped with 2 m H2SO4. The titre was determined as the double rate of the negative control.
Characterization of anti-N. bombycis monoclonal antibodies
Isotyping
The analysis of the mAb IgG subtype was performed following the protocol of SBA ClonotypingTM System/HRP (Southern Biotechnology Associates, Inc., Birmingham, AL35260, USA).
Cross-reactivity tests
The reactivity of antibodies with bacteria (Enterococcus faecalis), yeasts (Hansenula polymorpha), and another microsporidium (Endoreticulatus-like microsporidium) was assessed by ELISA.
Effects of anti-N. bombycis mAbs on microsporidial germination in vitro
In order to determine the linearity between the concentration of N. bombycis spores and absorbance at 625 nm, purified N. bombycis spore suspensions from 0 to 32 μl in 0·5 μl increments were brought to 150 μl by adding distilled water. The OD625 was measured on a spectrophotometer (BECKMAN DU 640). A 10 μl aliquot of every third dilution (0·5, 2·0, 3·5 μl, etc.) was examined on a Neubauer counting chamber under phase-contrast microscopy to determine spore concentration. The remaining spore concentrations were derived according to dilution ratio calculation. Microsoft Excel 2000 software was used to correlate the spore concentration to absorbance (OD625).
The effects of anti-N. bombycis mAbs on microsporidial germination in vitro were determined by incubating 20 μl of the purified N. bombycis spores (5×107 sp/ml) with 100 μl each of mAbs 3C2, 1A6, 3B1, 3C3, 3C4, 3F1 (1 μg/ml, 5 μg/ml, 50 μg/ml, 100 μg/ml, 500 μg/ml). An irrelevant isotype, the anti-infectious flacherie virus (IFV) Bombyx mori mAb 3E12 was used as a negative control. After washing to remove unbound antibodies, the spores were incubated at 37°C in 400 μl of GKK germination medium (0·05 mol/l glycine, 0·05 mol/l KOH, 0·375 mol/l KCl, pH 10·5) for 1 h (Undeen and Avery, Reference Undeen and Avery1984). The ungerminated spores showed the maximum absorbance at a wavelength of 625 nm (Undeen and Avery, Reference Undeen and Avery1988). As a dilution and blank control, 20 μl of spores were washed and incubated in 400 μl of H3PO4 medium (0·02 ml/l H3PO4, pH 6·0) or 400 μl of GKK.
Effects of mAb 3C2 on N. bombycis infecting the BmN cells in vitro
Twenty μl of purified spore suspension (5×107 sp/ml) and 200 μl of KOH (0·2 mol/l, pH 11·0) were first incubated for 1 h at 25–27°C, then with 200 μl of 1·0 mg/ml mAb at 37°C for 1 h. After washing to remove unbound antibody, spores were plated in 8-well plates containing a sterile cover-slip in the bottom of each well along with BmN cell cultures. BmN cell cultures were maintained with supplemented TC-100 medium (GIBCO-BRL). DAPI (Sigma) stained cover-slips were observed at 12, 24, 36, 48, 60, 72, 84 and 96 h post-infection under epifluorescent microscopy.
The influence of continuous exposure to mAb 3C2 on N. bombycis reproduction in vitro was determined by maintaining N. bombycis-infected BmN cells with medium containing 5 μg/ml, 50 μg/ml, or 500 μg/ml of mAb 3C2. An irrelevant isotype (anti-IFV mAb 3E12) at the same concentrations was used as a negative control. Cover-slips were harvested at 12, 24, 36, 48, 60, 72, 84 and 96 h post-culture and stained with DAPI. Spores within BmN cells were counted using an Olympus epifluorescent microscope and the percentage infected cells/field was determined.
Gel electrophoresis of proteins and immunoblot analysis
For 2-dimensional electrophoresis, spore proteins were extracted in a buffer containing 8 m urea, 40 mm CHAPS and 5% 2-mercaptoethanol. Isoelectric focusing (IEF) was carried out at 400 V for 4 h, 600 V for 30 min and 800 V for 30 min using the following ampholyte combination: 40% pH 3–10, 60% pH 4–6·5 (Pharmalyte, Pharmacia Biotech). After equilibration of IEF gels in 2% SDS, 5% 2-mercaptoethanol for 10 min, second-dimension SDS-PAGE was carried out on 12% polyacrylamide gels. Proteins were silver stained. For Western blot immunodetection, proteins were electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon P, Polylabo). Non-specific binding sites were blocked in PBS-5% skimmed milk for 0·5 h at room temperature. After incubation for 3 h with 1:100–1:5000 dilutions of mice antisera in PBS-0·1% Tween 20 and several washes in PBS-0·5% milk-0·1% Tween 20, the membranes were treated with a 1:5000 dilution of alkaline phosphatase-conjugated goat anti-mouse IgG (or IgM) (Sigma) for 1 h at room temperature. Blots were detected using substrates of NBT and BCIP. As a negative control, the anti-IFV mAb 3E12 was used.
Monoclonal antibody-based immunogold electron microscopy
Microsporidian spores fixed in 0·1 m sodium phosphate buffer containing 3% paraformaldehyde and 1% glutaraldehyde were embedded in LR white resin. Ultrathin sections on copper grids coated with formvar were incubated with appropriate ascites and then with a 1:100 dilution of gold-labelled goat anti-mouse IgG (Goat A MigG/gold/10 nm). The anti- IFV mAb 3E12 was used as a negative control. The grids were stained with uranyl acetate and incubated with lead citrate (Sak et al. Reference Sak, Saková and Ditrich2004). The samples were examined in a JEOL 1200EX transmission electron microscope.
Statistical analysis
Statistically significant differences were measured using the two-tailed Student's t-test by Statistical Package for Social Science (SPSS version 12.0; SPSS Inc., Chicago, IL). All experiments were performed in triplicate with 3 samples per experiment. Experimental data were compared with corresponding controls and P values of 0·05 or less were considered significant.
RESULTS
Generation of anti-N. bombycis monoclonal antibodies
Of 352 derived hybridomas, 31 reacted positively with N. bombycis antigen. The 7 most strongly reacting hybridomas were selected and cloned 3 times by limiting dilution. A total of 121 subclones were obtained of which 7 (named 1A6, 3B1, 3C1, 3C2, 3C3, 3C4, 3F1) showed positive reaction to N. bombycis antigen. These monoclonal antibody-producing hybridomas were multiplied in ascites, mAbs were purified from the ascites, and protein concentrations were measured.
Characterization of derived mAbs
Isotype
All 7 mAbs provided lines in radial immunodiffusion with anti-isotype sera, and all of them showed an anti-IgG line, 5 of them (3B1, 3C1, 3C2, 3C3, 3F1) had the dominant line against anti-IgG2 subtype and 1A6, 3C4 against anti-IgG1 subtype.
Specificity
ELISA showed that only 1A6 strongly cross-reacted with the Endoreticulatus-like microsporidium, while the others did not cross-react with this microsporidium. No other cross-reactions were observed.
MAb 3C2 inhibited the germination and reproduction of N. bombycis spores in vitro
Effects of mAbs on N. bombycis germination in vitro
N. bombycis spores darkened upon germination as seen under phase-contrast microscopy. The process was accompanied by a loss of optical density (OD) that was monitored spectrophotometrically permitting study of the kinetics of germination (Undeen and Avery, Reference Undeen and Avery1988). N. bombycis spore concentration was highly correlated with OD625 with a linear relationship of Y=2·712×106X (R=0·998) (Fig. 1).
Fig. 1. The relationship between Nosema bombycis spore concentrations (haemocytometer counts) and optical density at 625 nm.
There was a considerable reduction of the number of germinated spores when the spores were pre-incubated for 24 h with mAb 3C2 and 1A6 at different concentrations (Fig. 2) compared with spores pre-incubated with an irrelevant isotype mAb at the same concentrations (P<0·01). 3C4 showed a similar effect (P<0·05). The other mAbs did not strongly affect the germination of spores (P>0·05).
Fig. 2. mAb 3C2, 1A6, 3B1, 3C1, 3C3, 3C4, 3F1-mediated inhibition of Nosema bombycis germination in vitro at different concentrations of the mAbs. Asterisks indicate significant differences.
Pre-exposure of spores to mAb 3C2 inhibited N. bombycis infecting cells
MAb 3C2, which produced the strongest inhibition of N. bombycis spore germination was selected for this study in which 1×106 spores were cultured with 1 mg/ml of either mAb 3C2 or isotype control mAb. Pre-treatment of spores with mAb 3C2 for 1 h reduced the amount of infected cells up to 48·6% (72 h post-inoculation) compared with isotype control mAb 3E12 (P<0·01) (Figs 3 and 4).
Fig. 3. Nosema bombycis cultures stained with DAPI after pre-treatment with 1·0 mg/ml mAb 3C2(A) or isotype control mAb. Cells were harvested 72 h post-culture, fixed, stained and examined at 1000× under epifluorescent microscopy. N, nucleus; C, cytoplasm; SP, sporoplasms.
Fig. 4. Percentage Nosema bombycis-infected BmN cells following incubation of spores with monoclonal antibodies 3C2 or isotype 3E12 (control). Asterisks indicate significant differences.
MAb 3C2 in culture decreases N. bombycis reproduction in vitro
To determine whether continuous exposure of N. bombycis to anti-exospore mAb 3C2 would affect microsporidial reproduction, N. bombycis/BmN cell cultures were maintained in media containing either mAb 3C2 or control isotype mAb at different concentrations. Cultures were fixed and stained with DAPI after inoculation. The number of infected cells was significantly lower in mAb 3C2-treated cultures compared to control isotype mAb-treated cultures (P<0·01) from 12 to 96 h post-culture (Fig. 5).
Fig. 5. Percentage of Nosema bombycis-infected BmN cells maintained in the presence of antibodies mAb 3C2 or isotype 3E12 (control) at different concentrations and times. Asterisks indicate significant differences.
When cells were maintained with 500 μg/ml mAb 3C2 or 500 μg/ml control isotype mAb 3E12, there was a significant decrease in the percentage of infected cells in cultures maintained in 500 μg/ml mAb 3C2 when compared to the control isotype mAb (P<0·01). This decrease was similar to results for a mAb 3C2 concentration of 5 μg/ml (data not shown).
Antigen determination-size and pI
The spore proteins were subjected to Western blotting after 2-dimensional gel electrophoresis (Fig. 6). The 84 kDa component recognized by mAb 3C2 corresponded to a prominent protein spot with pI close to 7·2 in the 2D gel. The test included anti-IFV monoclonal antibody 3E12 as a negative control and this mAb yielded no visible reaction with N. bombycis antigens.
Fig. 6. Immunoreactivity of an 84 kDa Nosema bombycis spore surface protein. 2D PAGE separated proteins were either silver stained (A) or transferred to PVDF membranes and incubated with mAb 3C2 directed against the 84 kDa spot (B). The reactive spot is faintly silver stained (arrow). Molecular sizes (kDa) and isoelectric points (3–10) are indicated.
Antigen determination-localization
The immunogold reaction showed that mAb 3C2 bound exclusively to the exospore. The reaction was specific and mAb 3C2 demonstrated an indistinct generalized localization to antigens (Fig. 7). There was no immunogold reaction in the control sample using mAb 3E12 (data not shown).
Fig. 7. Immunogold electron micrograph of an section through Nosema bombycis spore immunostained with 3C2 mAb and labelled with 10 nm gold particles (arrows). Ex, exospore; En, endospore; PT, polar tube.
DISCUSSION
Spore germination in N. algerae and other microsporidia ends with darkening of the spore as observed under phase-contrast microscopy. This process is accompanied by a loss of optical density in a spore suspension and can be monitored spectrophotometrically to study the kinetics of germination (Undeen and Avery, Reference Undeen and Avery1988). N. bombycis spores also become phase-contrast dark upon germination and the process is accompanied by the expulsion of the polar tube and the sporoplasm. The positive correlation between OD reduction and percentage germination shows that spectrophotometry is a feasible method for measuring spore germination, and OD measurement is probably a more efficient method for estimating percentage germination than differential spore counts because a much greater number of spores can be sampled.
Continuous culture of N. bombycis with the monoclonal antibody 3C2 resulted in the reduction of germination rate and reproduction in BmN cells in vitro. Because only spectrophotometry (3100) and epiflorescent microscopy were used for the evaluation of experiments, we could not determine whether mAb 3C2 inhibited infectivity or overall microsporidial reproduction. However, the mean number of germinated spores and intracellular spores was significantly lower for 3C2 mAb-treated cultures than for control cultures (P<0·05). Thus, the invasion rate of the spores appears to have been affected by mAb 3C2. A partial role of specific mAbs in controlling microsporidiosis was documented by Enriquez et al. (Reference Enriquez, Wagner, Fragoso and Ditrich1998). The authors observed that pre-incubation or continuous culture of Encephalitozoon with their pan-specific anti-exospore mAb 3B6 also resulted, to some extent, in reduction of Encephalitozoon spp. reproduction in vitro. To reduce the error, we used an irrelevant mAb isotype as a control and, in addition, a GKK medium control and a H3PO4 medium as blank control.
The derived mAb 3C2 reacted with IgG anti-isotype sera line anti-IgG2 subtype. The ELISA revealed that only 1A6 strongly cross-reacted with an Endoreticulatus-like microsporidium, while mAb 3C2 did not cross-react with the Endoreticulatus-like microsporidium. The cross-reaction of anti-N. bombycis mAbs with Endoreticulatus-like microsporidium spores was not surprising due to their close phylogenetic relationship (Baker et al. Reference Baker, Vossbrinck, Becnel and Andreadis1998). Enriquez et al. (Reference Enriquez, Ditrich, Palting and Smith1997) also developed a mAb against E. cuniculi that cross-reacted with antigens of E. intestinalis and E. hellem. Sak et al. (Reference Sak, Saková and Ditrich2004) developed mAbs against E. cuniculi that cross-reacted with antigens of E. intestinalis. It is possible that the antigenic determinants recognized by 1A6 mAb are highly conserved between Nosema and Endoreticulatus spp. 3C2 reacted with antigens of the spore wall and demonstrated a generalized localization to exospore antigens. The mAb 3B6 against exospore antigens developed by Enriquez et al. (Reference Enriquez, Ditrich, Palting and Smith1997) recognized immunoblot bands of 34, 40, 46, 53, 63, 102 and 117 kDa; similarly Sak et al. (Reference Sak, Saková and Ditrich2004) developed mAb P5/H1 against exospore antigens that recognized 2 bands of E. cuniculi antigens, but they were very close in size to those observed by Enriquez (50 and 98 kDa). Our mAb 3C2 reacted only with an 84 kDa protein corresponding to pI (7·2) on the 2D gel. Such a protein may be of interest for diagnostic purposes because of its strong antigenicity in immunoblotting assays with various antisera collected from microsporidia-infected mice or rabbits.
Although the mechanism by which the 3C2 mAb inhibits the germination and invasion of N. bombycis in vitro remains to be determined, several possibilities may warrant exploration, such as its effects on limitation or exacerbation of the spores' extrusion of the coiled polar filament. Another explanation for the reduction of the germinated spores in vitro may be that 3C2 targeted neutralization-sensitive epitopes in N. bombycis spores. The ability of spores to germinate may be impaired when antibody is bound to the exospore. Monovalent ions are essential for in vitro germination of N. bombycis (Weidner, Reference Weidner and Byrd1982). Dall (Reference Dall1983) suggested that alkaline conditions establish a proton gradient across the spore plasma membrane, which facilitates the activation of ionosphere molecules in the spore membrane. This proton gradient drives the cation/proton exchange across the membrane and alkalinity is developed within the spore that, in turn, drives cation proton exchange between organelles such as the polaroplast and the sporoplasm. The combined transport of those ions may cause an osmotic imbalance, leading to the influx of water and discharge of the polar filament and sporoplasm (Dall, Reference Dall1983). It may be speculated that the epitopes correlate to the ion channels and antibodies disturb the ion exchange resulting in fewer germinated spores. It is possible that the antigens are associated with aquaporin (AQP) CHIP28-like proteins because the flow of water into the spores is regulated by a specific trans-membrane pathway that involves acute sensitivity to Hg2+ (Frixione, Reference Frixione, Ruiz, Cerbon and Undeen1997). Ghosh (Reference Ghosh, Cappiello, McBride, Occi, Cali, Takvorian, McDonald and Weiss2006) cloned an AQP-like sequence of the microsporidium Encephalitozoon cuniculi (EcAQP). Aquaporin function is typically assayed in RNA-injected Xenopus oocytes, where swelling occurs under osmotic stress due to expression of the exogenous AQP (Verkman and Mitra, Reference Verkman and Mitra2000; Agre and Kozono, Reference Agre and Kozono2003). Utilizing this assay, the osmotic permeability and solute conductivity of EcAQP-injected oocytes were investigated (Ghosh, Reference Ghosh, Cappiello, McBride, Occi, Cali, Takvorian, McDonald and Weiss2006). In animals, these consist largely of ion channels with communication roles such as in signal transduction, or roles as sensors for external stimuli. Aquaporins may be important for explaining the sudden extrusion of the polar tube in relation to a water influx shown to occur through a specific transmembrane pathway.
Antibodies could play an important role in the control of microporidiosis, particularly those targeting epitopes on exospore antigens. The presence of an antibody stimulates the immune response, making phagocytosis more efficient and increasing the oxidative burst of murine macrophages (Sak et al. Reference Sak, Saková and Ditrich2004). This mechanism, together with the antibody-dependent neutralization of sensitive epitopes in microsporidia, may be a part of the host strategy to control infections. From the present results it can be concluded that the germination of microsporidian spores is influenced by the surface proteins of spores. When the surface proteins of spores are restrained or destroyed, the germination and invasion of host cells will be effectively inhibited. The spore surface proteins may play an important role in the parasitizing cells and further studies should lead to development of novel strategies for control of these important parasites.
This research was supported by the Natural Science Foundation of China (Project No: 30270898).
