INTRODUCTION
Apicomplexan parasites cause some of the world's most debilitating diseases including malaria and toxoplasmosis in humans, babesiosis and theileriosis in cattle and coccidiosis in chickens. In the case of the Plasmodium parasite, malaria is still one of the world's biggest killers leading to the death in 2010 of more than half a million people, most of these deaths occurring in young African children (World Health Organization, 2010). Eimeria, the causative agent of coccidiosis in chickens and other animals, has had a major economic impact on the meat industry and, in spite of 70 years of drug development, is still difficult to control. Most of these parasites undergo complex life cycles going through both asexual and sexual stages of development, leading to the production of oocysts that act to transmit the parasite in either an insect vector, as is the case for malaria, or the environment as in the case of Coccidia. Coccidian oocysts remain viable under hostile conditions due to the presence of an oocyst wall; a structure that enables parasite survival while preventing the influx of toxic substances.
A great deal of research has focused on elucidating the structure, biochemical composition and developmental biology of the oocyst wall of Eimeria spp. Eimeria maxima is an apicomplexan parasite that is 1 of the 3 major species of Eimeria that cause coccidiosis in chickens. In situ studies using fluorescent microscopy to observe sexual stage development in tissue sections of chicken intestines infected with E. maxima, revealed that the 2 types of wall-forming bodies (WFBs), Type 1 and 2 (WFB1s and WFB2s), migrate to the periphery of the parasite, undergo fusion and form the outer and inner layer of the wall respectively (Mehlhorn, Reference Mehlhorn1971, Reference Mehlhorn1972; Pittilo and Ball, Reference Pittilo and Ball1979, Reference Pittilo and Ball1980; Ferguson et al. Reference Ferguson, Belli, Smith and Wallach2003).
In previous studies it was found that a collection of gametocyte antigens, termed Affinity Purified Gametocyte Antigen (APGA), prepared using a monoclonal antibody to the gametocyte 56 kDa antigen, elicit a protective immune response against parasite transmission. APGA has been used as the basis of a commercial transmission-blocking vaccine against coccidiosis, CoxAbic® (Wallach et al. Reference Wallach, Halabi, Pillemer, Sarshalom, Mencher, Gilad, Bendheim, Danforth and Augustine1992, Reference Wallach, Ashash, Michael and Smith2008; Smith et al. Reference Smith, Wallach, Petracca, Braun and Eckert1994). The 56 and 82 kDa gametocyte antigens contained in APGA and their peptide fragments have been localized to the WFB2, and have been shown to compose the inner layer of the oocyst wall (Ferguson et al. Reference Ferguson, Belli, Smith and Wallach2003). Furthermore, the genes encoding these antigens have been cloned and sequenced where it was found that they are very rich in the amino acid tyrosine (Belli et al. Reference Belli, Witcombe, Wallach and Smith2002b). Based on the finding that when zygotes and oocysts of E. maxima are exposed to UV light they autofluoresce blue, work was carried out in which it was shown that during oocyst wall formation these proteins are processed into smaller peptides, enzymatically cross-linked via the formation of di-tyrosine (the molecule that fluoresces blue) and inserted into the inner oocyst wall (Belli et al. Reference Belli, Wallach, Luxford, Davies and Smith2003; Mai et al. Reference Mai, Sharman, Walker, Katrib, De Souza, McConville, Wallach, Belli, Ferguson and Smith2009, Reference Mai, Smith, Feng, Katrib, Slapeta, Slapetova, Wallach, Luxford, Davies, Zhang, Norton and Belli2011).
In spite of all of the above research, little is known about the molecular composition and biosynthesis of the WFB1s and the molecular mechanisms underlying outer oocyst wall formation. In addition, the proteomic analysis of the 2 types of WFBs is still incomplete. Therefore, in the present study confocal and wide-field fluorescent microscopy combined with a variety of staining techniques, were used for the first time to observe in 3 dimensions the subcellular location and spatial organization of the WFBs at various stages of parasite development in isolated and purified gametocytes. In addition, it was shown that extracted WFBs had the same size, shape and staining properties as in situ WFBs of macrogametocytes extracted from the E. maxima-infected intestine, and contained the expected glycoproteins previously shown to be present in these organelles. This work therefore is the first step in the purification of the WFBs that can then be used for future biochemical and proteomic analysis.
MATERIALS AND METHODS
Oocyst, gametocyte and enterocyte production
The Houghton strain of E. maxima was originally provided by Dr Martin Shirley (Institute for Animal Health, Compton, Newbury, Berkshire, UK). Oocysts were routinely passaged through 4-week-old chickens (Australorps; S. F. Barter and Sons) and purified as described previously (Belli et al. Reference Belli, Lee, Thebo, Wallach, Schwartsburd and Smith2002a). Fresh oocysts were used to infect chickens and macro- and microgametocytes, which after concentration on a 10 μm polymon filter, were then purified from infected intestines 134 h post-infection (h p.i.) as described previously (Pugatsch et al. Reference Pugatsch, Mencher and Wallach1989). The harvested macrogametocytes were then assessed for the presence of WFBs by cytochemical stains, Western blotting and immunofluorescence microscopy using WFB-specific antibodies, as described below.
For chicken enterocyte preparation, a total of 5 uninfected Australorp cockerels were euthanized at 4 weeks of age and their intestines removed and immediately washed with ice-cold phosphate-buffered saline (PBS, pH 7), slit open and cut into small pieces measuring approximately 1 cm2. Tissue was then incubated with 0·5 mg mL−1 hyaluronidase in PBS (pH 7) and placed in a 41 °C water bath for 30 min. At the end of the incubation period the intestines were filtered through cheesecloth and the material retained on the cheesecloth was discarded and the flowthrough, containing the enterocytes, was filtered through a 10 μm polymon filter (Sefar Filter Specialist Pty Ltd). The enterocytes, which pass through the filter, were collected and washed 3 times by centrifugation at 1000 g for 10 min. The enterocytes were then examined microscopically and diluted to 2 × 106 mL−1 and stored at −20 °C until needed. Staining with haematoxylin and eosin (H&E) was performed to check the quality of the harvested cells.
Live cell imaging of harvested E. maxima gametocytes
Freshly harvested gametocytes re-suspended in 200 μL of TNEP buffer (10 mm Tris-HCl, pH 7·4, 50 mm NaCl, 2 mm EDTA) were placed on 0·1% (v/v) poly-L-lysine-coated glass microscope slides before imaging in a microscope heated chamber warmed to 37 °C. Images were captured with a high-speed charge-coupled device (CCD) camera using the NIS-Elements acquisition software mounted on a Nikon Ti inverted microscope equipped with 60× oil objective lens (Plan Apo NA 1.4 aperture) and the Perfect Focus System™ for continuous maintenance of focus. Blue autofluorescence was monitored with a bandpass excitation/emission filter (330–385/435–485 nm). Time-lapse images were collected every 30 min over a 17 h period. Exposure time and brightness/contrast settings were kept constant for each using the NIS Elements acquisition software.
Tissue processing and H&E staining of in situ and isolated gametocytes
Portions of the small intestine containing E. maxima gametocytes were fixed in 10% neutral buffered formalin (pH 7). Fixed tissues were embedded in paraffin, 5 μm thick sections were cut and stained with H&E at the Histopathology Laboratory (Faculty of Veterinary Science, University of Sydney, New South Wales, Australia). Colour images were acquired with the Olympus BX51 upright epifluorescence microscope equipped with a DP70 Charged Couple Device (CCD) Microscope Camera.
Freshly harvested gametocytes were placed on glass slides pre-coated with 0·1% poly-L-lysine (Sigma) for 30 min, at room temperature. The unbound material was washed off and the slides air-dried and fixed in 95% ice-cold methanol for 5 s or left unfixed, and the organelle composition of preparations was determined by staining with H&E, nile red and oil red O, or alternatively, probed with antibodies as described below.
H&E staining was carried out on unfixed parasites using a modified staining protocol. Briefly, the samples were soaked in Harris haematoxylin for 1 min; rinsed in tap water; treated with 1% acid alcohol, and then rinsed again with tap water for 10 s. The samples were then soaked in Scott's water for 1 min; rinsed; treated with 1% alcoholic eosin for 1 min, and de-stained twice in 95% ethanol for 30 s. Samples were dehydrated in 100% ethanol for 1 min, and submerged in xylene; 2 changes at 2 min intervals. Once dehydrated, the samples were mounted under cover slips in D.P.X. mounting medium and visualized under bright field using an Olympus BX51 upright microscope with 100× objective.
Indirect immunofluorescence analysis
Gametocytes and extracted WFBs were fixed for 5 min at room temperature in 95% ice-cold methanol, permeabilized in 0·1% TWEEN20/PBS and non-specific staining was blocked with 2% BSA in PBS for 1 h, at room temperature. The harvested gametocytes were then incubated with either a mouse monoclonal anti-WFB1 antibody E1D8, raised against an antigen in the WFB1 of E. tenella gametocytes (Mouafo et al. Reference Mouafo, Weck-Heimann, Dubremetz and Entzeroth2002), or incubated with a mouse monoclonal anti-WFB2 antibody (1 : 200 dilution) raised against E. maxima gametocyte glycoprotein EmGAM56 (Fried et al. Reference Fried, Mencher, Sarshalom and Wallach1992) and counterstained with diamidino-2-phenylindole (DAPI). Primary antibodies were detected with Alexa Fluor 488 – conjugated goat anti-mouse IgM (1 : 500 dilution, Invitrogen, Australia). Normal chicken and mouse sera, or isotype-matched purified mouse myeloma IgM, kappa antibody (Sigma), were used as a negative control. Localization of bound antibodies was visualized with an Olympus BX51 upright epifluorescence microscope with a bandpass excitation/emission filter (470–490/510–550 nm). Multinucleated microgametes stained with DAPI were examined with a Nikon A1 Confocal Scanning Laser Microscope equipped with a 100× oil immersion objective lens (N.A. 1.4) and a violet laser (405/425–475 nm). The three-dimensional volumes of blue fluorescing microgametes were then deconvolved using AutoQuant deconvolution software (7.5.1 Bitplane, Zürich, Switzerland) and displayed as maximum z-projections.
Labelling of E. maxima gametocytes with nile red and oil red O
The lipid content of the WFBs was examined by nile red staining according to the method of Greenspan et al. (Reference Greenspan, Mayer and Fowler1985). For the detection of both brilliant yellow gold fluorescence typical of neutral lipids (cholesterol and triglycerides), and red fluorescence characteristic of glycoproteins, phospholipids, amphipathic lipids and strongly hydrophobic membrane proteins, cells were examined with an Olympus BX51 upright microscope (Olympus, Australia) using 100× oil immersion objective (N.A. 1.3) and a DP70 colour CCD camera (Olympus Australia Pty Ltd). Nile red-stained red and yellow emission was observed with 460–490 nm excitation and 510 nm long-pass emission filters. In order to achieve a three-dimensional visualization the subcellular location and distribution of lipids/glycoproteins in the membranes/walls surrounding the developing zygotes/oocysts, a Nikon A1 Confocal Scanning Laser Microscope equipped with a 100× oil immersion objective lens (N.A. 1.4) was employed. Cells stained with nile red were excited with the 488 nm laser and yellow and red emissions were observed with 525/50 nm and 595/50 nm emission filters, respectively. To acquire three-dimensional volumes, 106-z serial optical sections were collected with a step size of 0·2 μm, using NIS Elements software (Nikon). Images were deconvolved using AutoQuant blind deconvolution software (7.5.1 Bitplane, Zürich, Switzerland) and displayed as maximum z-projections.
Oil red O staining was performed as described previously (Pittenger et al. Reference Pittenger, Mackay, Beck, Jaiswal, Douglas, Mosca, Moorman, Simonetti, Craig and Marshak1999). Briefly, harvested gametocytes were covered with 60% isopropanol in PBS (pH 7) for 2–5 min at room temperature. Isopropanol was poured off and 20 μL of oil red O dye was added to each sample for 5 min. After incubation, slides were washed in distilled water for 5 min, and counter-stained in Mayer's Haematoxylin and mounted in D.P.X. (Fronine, Australia) prior to visualization under bright field, as described above.
Extraction of gametocyte WFBs
Purified E. maxima gametocytes (2 × 106 cells mL−1) were re-suspended in TNEP (10 mm Tris-HCl, pH 7·4, 50 mm NaCl, 2 mm EDTA) buffer and mechanically disrupted by sonication (Vibra Cell Sonicator) using a 2-mm microtip and a wave amplitude of 2·5 W for consecutive 10-s intervals over 0.5 min. To remove released RNA, DNAse-free RNAse A (Sigma) was added to the lysate at a final concentration of 10 μg mL−1, according to the manufacturer's instructions. The sonicate was then centrifuged at 15 000 g for 10 min and the pellet was re-suspended in 1% sodium-dodecyl-sulphate (w/v) to separate the insoluble material from solubilized debris. The detergent supernatant was then passed through a 1000 kDa cut-off filter (MWCO, VivaSpin) and washed three times in TNEP (pH 7·4) by centrifugation (15 000 g for 5 min). Fractions were collected and the protein content of each fraction was estimated using the bicinchoninic protein assay (Pierce), according to the manufacturer's instructions and using bovine serum albumin as a standard. All enriched fractions were assessed for the presence of WFBs by cytochemical stains, immunofluorescence microscopy and Western blotting using WFB-specific antibodies.
SDS–PAGE and immunoblotting
The proteins from gametocyte lysates, enriched fractions and host cells were separated by size as described previously (Belli et al. Reference Belli, Lee, Thebo, Wallach, Schwartsburd and Smith2002a). Laemmli sample buffer was added to all fractions prior to boiling for 10 min at 95 °C. Samples were then centrifuged at 12 000 g for 5 min, at room temperature to remove insoluble and aggregated matter prior to SDS–PAGE on NuPAGE® Novex 4–12% Bis-Tris gradient gels (Invitrogen). Total protein (4 μg) loaded per lane was determined by bicinchoninic protein assay (Pierce). The proteins were then visualized by staining with silver or, alternatively, transferred to polyvinylidene difluoride (PVDF, Pall Corporation) membranes and incubated with mouse anti-1E11–11 monoclonal antibody or anti-APGA at a 1 : 1000 dilution (Wallach et al. Reference Wallach, Pillemer, Yarus, Halabi, Pugatsch and Mencher1990), or normal serum (1 : 1000). PVDF membranes were then incubated with alkaline-phosphatase conjugated rabbit anti-mouse IgM or anti-chicken IgG (1 : 1000; Sigma) and developed with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (SIGMA FAST ™ BCI/NBT, Sigma) according to the manufacturer's instructions.
RESULTS
Isolated E. maxima macrogametocytes continue to develop in vitro and contain intact WFBs
To visualize autofluorescence in isolated macrogametocytes, time-lapse video microscopy of developing parasites was used to observe UV-excited fluorescence. Sporulated oocysts, which were used as an internal control for autofluorescence, were added to the gametocyte sample. During the 17-h time-period, the isolated macrogametocytes increased in autofluorescence while, simultaneously, the WFBs became detectable at the periphery of the parasite (Fig. 1) whilst the sporulated oocyst wall showed no increase in fluorescent intensity. These results indicated that macrogametocyte maturation continues even after E. maxima macrogametocytes are removed from the infected intestine.
Time-lapse live cell imaging of freshly harvested Eimeria maxima macrogametocytes. The maturation of freshly isolated macrogametocytes is revealed by an increase in autofluorescence over a 17 h period. Sporulated oocysts were used as a reference for autofluorescence. In each series, the images on the left-hand side represent the phase-contrast micrographs and the images on the right-hand side show autofluorescence at 330–385 nm excitation wavelength. Bar = 20 μm.
To visualize the WFBs and gain further insights into oocyst wall formation, macrogametocytes at various stages of maturation were examined by H&E staining. Microscopic analysis of gametocytes purified 134 h post-infection from the infected intestine revealed that all stages of sexual development are present (Fig. 2A–K), consistent with previous reports that 134 h is the peak time-point for gametocyte production (Ferguson et al. Reference Ferguson, Belli, Smith and Wallach2003). The early macrogametocytes, which were identified by their single centrally located nucleus, appeared to contain a number of eosinophilic bodies of approximately 0·5 to 1 μm in diameter (Fig. 2A and B, arrows). These eosinophilic bodies appeared to extend outward from the nucleus and were therefore identified as early macrogametocyte WFBs. In addition, numerous basophilic granules appeared dispersed in the cytoplasm of an early macrogametocyte (Fig. 2A and B, arrowheads). These granules reached a diameter of 0·4 ± 0·1 μm and, based on their appearance and distribution, were identified as veil-forming bodies (VFBs) (Fig. 2A and B, arrowheads). At this stage, using the H&E dye it was not possible to detect the ‘doughnut-shaped’ granules, characteristic of the WFB2s in the cytoplasm of the isolated macrogametocytes. However, mature macrogametocytes, which were larger in size, possessed large WFB1s, which appeared in the peripheral cytoplasm with narrow bridges linking them together into chains (Fig. 2C and D). These WFB1s reached a maximum 2·5 μm in diameter and appeared to be coated by a membranous basophilic structure and were cross-linked via eosinophilic ‘bridges’ extending laterally from each organelle (Fig. 2D). In some specimens, we observed the coalescence of the WFB1s to form the thick eosinophilic outer oocyst wall (Fig. 2E and F). In newly formed oocysts, the thin basophilic layer, presumably the inner layer of the oocyst wall was detected directly underneath the thick outer oocyst wall (Fig. 2G and H).
Light microscopy of freshly harvested (A–K) and in situ (L–O) macro- and micro-gametocytes stained with H&E. (A) An early harvested macrogametocyte showing a single basophilic centrally located nucleus with eosinophilic and basophilic bodies dispersed in the cytoplasm. (B) Detail of a part of the isolated macrogametocyte showing eosinophilic granules (arrows), presumably early WFBs concentrically arranged around the nucleus (Nu). Note the presence of numerous basophilic bodies, resembling the VFBs (arrowheads). (C) A mature isolated macrogametocyte showing large Type 1 WFBs (WFB1) arranged into necklace-like structures in the peripheral cytoplasm. (D) Enlargement of the section shown in (C), illustrating the membrane surrounding the WFB Type 1 organelles (arrows) and their arrangement into ‘bunch of grapes’ with narrow bridges linking them together. (E, F) A forming oocyst showing large WFB1s (arrows) fusing with the parasite's limiting membrane. (G, H) A newly formed oocyst showing 3 distinct layers, including a basophilic loose outermost layer, presumably the veil, a thick eosinophilic outer oocyst wall (OW) and a thin basophilic inner layer (IW). (I–K) Microgametocytes isolated 134 h p.i. at various stages of maturation revealed with H&E stain. (I) An early microgametocyte showing peripheral basophilic nuclei (Nu) approximately 7 μm in diameter and a number of undifferentiated early microgametes (EMi). An oocyst (O) attached to the surface of a microgametocyte. (J) A mature microgametocyte with fully formed microgametes (MMi), each reaching 5–7 μm in length and containing a small basophilic nucleus. Note the swollen host nucleus protruding from the host cell. (K-k) A late-stage microgametocyte (LMi), showing protrusion of fully formed microgametes into the exterior (arrows). Mature microgametes appear comma-shaped, each containing a posterior basophilic nucleus (k). (L–O) Macrogametocytogenesis of Eimeria maxima in tissue sections of infected chicken intestine. (L) An early in situ macrogametocyte situated in the parasitophorous vacuole (PV) showing a basophilic nucleus (Nu) and a number of small eosinophilic early WFBs. (M) A mature macrogametocyte showing large, membrane-bound eosinophilic WFB1s in the peripheral cytoplasm and eosinophilic masses, the aggregated WFB2s. (N) An early oocyst showing a number of coalescing WFB2s (arrows) underneath the outer OW. (O) Formed oocyst. Bar corresponds to 5 μm. Abbreviations: HN, host nucleus; Emi, early microgametocyte; Mi, microgametocyte; MMi, mature microgametocyte; LMi, late microgametocyte; Nu, nucleus; O, oocyst; OW, outer oocyst wall; PV, parasitophorous vacuole; WFB1, Type 1 wall-forming bodies; WFB2, Type 2 wall-forming bodies.
In addition to macrogametocytes, a few microgametocytes at various stages of maturation were also observed. The early to mid-stage microgametocytes were identified as they lack WFBs, and instead were found to contain a number of large, basophilic peripherally located nuclei, typically 7 μm in diameter (Fig. 2I). Dark-stained granules, which concentrated at the periphery of these nuclei, were identified as chromatin. At this stage, the individual microgametes were barely detectable while in later stages of microgametocytogenesis large numbers of microgametes occupied a single host cell with the host nucleus swollen and pushed to the periphery (Fig. 2J). Microgametes, 5 to 7 μm in length, appeared comma-shaped and contained a small, posterior basophilic nucleus (Fig. 2K-k). Finally, in isolated microgametocytes we observed fully formed microgametes that protruded into the parasitophorous vacuole and were detached from the host cell (Fig. 2 K-k).
To further demonstrate that the WFBs observed in isolated gametocytes were intact, a comparison was made with WFBs seen in stained tissue sections (Fig. 2L–O). Serial thin sections of H&E-stained in situ macrogametocytes revealed a similar size, shape and distribution of WFBs to those seen in isolated gametocytes (Fig. 2A–H). The early gametocytes in tissue sections contained a single centrally located nucleus with distinguishable eosinophilic early WFBs arranged around it. Once again, it was difficult to discern Type 2 from Type 1 WFBs at this stage of macrogametocyte maturation (Fig. 2L). In contrast, the mature macrogametocytes contained 2 distinct populations of WFBs situated in the peripheral cytoplasm (Fig. 2M). The large, membrane-bound eosinophilic WFB1s were seen in the peripheral cytoplasm of the mature macrogametocyte, whereas the smaller eosinophilic WFB2s appeared aggregated, forming amorphous eosinophilic masses. Similar to WFB1s of isolated gametocytes, the membrane-bound WFB1s in tissue sections were situated near the parasite's surface, occasionally fused with the parasite's limiting membrane to form the thick, outer oocyst layer (Fig. 2N). The amorphous WFB2s aggregated into eosinophilic masses that formed the inner oocyst layer (Fig. 2N and O), consistent with a previous publication (Mehlhorn, Reference Mehlhorn1971).
To more specifically assess the presence of antigenic glycoproteins in the gametocyte WFBs, harvested gametocytes and/or zygotes were analysed by immunofluorescent microscopy. Monoclonal antibody 1E11-11, raised against the EmGAM56 glycoprotein localized to the ‘doughnut-shaped’ WFB2s dispersed in the cytoplasm of an early macrogametocyte (Fig. 3A and B), and the inner wall of the forming oocyst (Fig. 3C), consistent with a previous report (Ferguson et al. Reference Ferguson, Belli, Smith and Wallach2003). No reaction was detected when control serum was used (Fig. 3D). The WFB1s were detected with the monoclonal antibody, E1D8, that is specific to an antigen found in the WFB1s of E. tenella (Mouafo et al. Reference Mouafo, Weck-Heimann, Dubremetz and Entzeroth2002). This antibody reacted to the WFB1s of macrogametocytes (Fig. 3E), and material deposited on the parasite's surface (Fig. 3F), in agreement with a previous report (Frolich et al. Reference Frolich, Entzeroth and Wallach2012). Interestingly, E1D8 also bound to material within the zygote's cytoplasm even after the outer wall was clearly visible (Fig. 3F, double arrows). No reaction was detected when isotype-matched purified mouse myeloma IgM, kappa antibody (Sigma) was used as a control (Fig. 3G). In addition, DAPI staining revealed a number of multi-nucleated, large microgametocytes (Fig. 3H). Taken together, these results indicate that the isolated macrogametocytes were not harmed during isolation from the infected chicken intestine, both in terms of morphology, as well as in terms of the molecular and antigenic composition of the WFBs.
Distribution of Type 1 and Type 2 wall forming bodies during sexual-stage development in macrogametocytes isolated 134 h p.i. from the infected chicken intestine. (A–C) Fluorescent micrographs of isolated macrogametocytes probed with a monoclonal antibody raised to Eimeria maxima glycoprotein EmGAM56. (A) An early macrogametocyte showing numerous positively stained ‘doughnut-shaped’ WFB2s dispersed in the cytoplasm. (B) Shows the enlarged ‘doughnut-shaped’ WFB2s detected in A. (C) Shows an early oocyst in which numerous 1E11-11-positive granules (green) are accumulated at the surface of the parasite, representing the initiation of inner wall formation. (D) Normal mouse serum control. (E, F) Isolated macrogametocytes immune-labelled with anti-WFB1 antibody, E1D8, generated to a set of antigens localized in the WFB1 of E. tenella gametocytes (Mouafo et al. Reference Mouafo, Weck-Heimann, Dubremetz and Entzeroth2002). (E) A mature macrogametocyte showing numerous large E1D8-positive WFB1s (green). (F) An oocyst showing E1D8-positive outer oocyst wall and a few ‘left-over’ WFB1s underneath (double arrows). (G) Isotype-matched purified mouse myeloma IgM, kappa antibody (Sigma) control. (H) A three-dimensional confocal reconstruction of harvested microgametocytes stained with a nuclear stain, DAPI (blue). Note the presence of numerous nuclei in the cytoplasm of the microgametocyte. The bar represents 5 μm.
Use of nile red and oil red O to stain the WFBs of developing macrogametocytes
Nile red and oil red O lipophilic stains were used to stain WFBs during gametocyte development and oocyst wall formation (Fowler and Greenspan, Reference Fowler and Greenspan1985; Greenspan et al. Reference Greenspan, Mayer and Fowler1985). Nile red staining patterns were examined by fluorescence microscopy as described above. As can be seen, nile red revealed numerous ‘doughnut-shaped’ Type 2 WFBs intermixed with a number of small, brightly staining yellow-gold granules in the cytoplasm of an early macrogametocyte (Fig. 4A and B). At the mid-stage of development, the yellow-gold fluorescence characteristic of neutral lipids localized to the large WFB1s, which reached a maximum diameter of 2·5 μm. In comparison, the ‘doughnut-shaped’ WFB2s appeared to be 1·8 μm in diameter and orange/red in colour indicating the presence of glycoproteins, amphipathic and/or polar lipids (Fig. 4C and D).
Stage-specific localization of neutral lipids, polar lipids and glycoproteins in freshly harvested macrogametocytes with lipid stains nile red and oil red O. (A) A fluorescent micrograph showing numerous ‘doughnut-shaped’ WFB2s dispersed in the cytoplasm of an early macrogametocyte. (B) Enlargement of the enclosed area in (A), highlighting the ‘doughnut-shaped’ WFB2s intermixed with small brightly stained lipid granules (yellow) of various sizes. (C) A mature macrogametocyte showing localization of neutral lipids (yellow) to the large WFB1s and localization of polar lipids and/or glycoproteins (orange/red) to the ‘doughnut-shaped’ WFB2s aggregated in the cytoplasm. (D) Detail of a part of a mature macrogametocyte, shown in C. (E) An early oocyst showing coalescence of the lipid-rich WFB1s (yellow) at the parasite's surface. (F) Enlargement of the enclosed area displayed in (F) showing deposition of the yellow-gold Type 1 WFBs into the empty space between the 2 parasite-derived membranes representing initiation of outer wall formation (OW). Note the presence of orange/red fluorescing loose outermost veil (V) surrounding the early oocyst. (G) A later stage to that in panel E, showing a loose orange/red fluorescing veil (arrow) and a yellow-gold outer oocyst wall underneath (OW). (H) A few ‘left-over’ large yellow-gold WFB1s, located at the periphery, are seen directly underneath the thick yellow-gold outer oocyst wall (OW). (I) A 3-dimensional confocal microscopy image showing an isolated forming oocyst stained with nile red, highlighting lipid-rich patches coded green (double arrow) and the parasite veil (V) and plasma membranes coded red; overlapping lipid-rich, phospholipid- and/or glycoprotein-rich regions appear yellow. (J) Three-dimensional confocal reconstructions of through-focal optical sections showing coalescence and deposition of lipid-rich patches (coded green) between the 2 parasite-derived membranes (red) giving rise to the lipid-rich outer layer of the oocyst wall. (K, L) A fluorescent microscopy image showing localization of the lipids (yellow) to the thick outer oocyst wall (OW) and a polar body (PB) in the unbleached sporulated oocysts (K) and the localization of the lipids (yellow) to the polar body of the bleached sporulated oocyst (L). Note the absence of a yellow-fluorescing outer oocyst wall and a remaining red-fluorescing thin inner oocyst wall (IW) in panel L. (M–P) Light microscopy of the isolated gametocytes (M), early oocyst (N), sporulated unbleached (O) and bleached oocysts (P) stained with oil red O. Note the accumulation of the oil red O dye in the large WFB1s and the outer oocyst wall of immature and mature unbleached oocysts (arrows) and the absence of the dye from bleached oocysts. Scale bar = 5 μm. Abbreviations: IW, inner oocyst wall; Nu, parasite nucleus; PB, polar body; OW, outer oocyst wall; WFB1, Type 1 wall-forming bodies WFB2, Type 2 wall-forming bodies.
In mature macrogametocytes, a red fluorescing membraneous structure was present surrounding the parasite (Fig. 4E). At this stage, large yellow-gold WFB1s were situated just below the parasite's plasmalemma and formed the thick yellow-gold layer filling the empty space between the two parasite-derived membranes lying internal to the red fluorescing outermost membrane, presumably the veil (Fig. 4F). Using nile red stain, it appeared that not all yellow-gold WFB1s were completely consumed in this process, and in some instances they remained as large organelles in the cytoplasm of formed oocysts (Fig. 4G and H, arrows). Also in the cytoplasm of forming oocysts, an additional group of brightly staining bodies became apparent. This population of granules appears to consist mainly of very small, brightly staining bodies, which reached a maximum diameter of only a 100 nanometers and therefore were significantly smaller than the large WFB1s. These granules were predicted to be lipid droplets, as described in previous studies (Ferguson et al. Reference Ferguson, Belli, Smith and Wallach2003). According to the nile red staining, these small brightly stained lipid granules appeared to be the most abundant at the very late stages of oocyst biogenesis (Fig. 4G).
Finally, three-dimensional confocal microscopy was used to observe macrogametocytes at various stages of maturation. Three-dimensional confocal images of merged yellow and red emissions were acquired of an early oocyst. The image shows coalesced lipid-rich WFB1s coded yellow at the parasite's surface forming yellow lipid-rich patches initiating the beginning of the outer oocyst wall formation (Fig. 4I). The two-dimensional focal micrograph of the same specimen showed the accumulation and condensation of the yellow-fluorescing material at the parasite's membrane lying internal to the red fluorescing layer, presumably the veil (Fig. 4J). It was found that these yellow patches had formed in a scattered fashion on the surface of the macrogametocyte.
Upon completion of oocyst wall formation and sporulation it was found that the entire surface was coated in a yellow-gold fluorescing outer layer (Fig. 4K). Treatment of the sporulated oocysts with bleach, completely stripped off the thick yellow-fluorescing outer oocyst wall, leaving the thin red-fluorescing inner wall intact (Fig. 4L). This result further supports the contention that the outer oocyst wall, like the WFB1s, is mainly composed of neutral lipids (e.g. triglycerides and cholesterol). Similar subcellular localization and distribution of lipid-rich material was evident based on staining with oil red O, which specifically binds to lipids and lipoproteins (Fig. 4M–P).
Extraction, staining and protein analysis of the WFBs of E. maxima
An extraction process was developed in order to isolate and enrich for gametocyte WFBs (see Materials and Methods). The results of staining and immunofluorescence analysis of these enriched WFBs are shown in Fig. 5. As can be seen, organelles that appeared purple-pink after H&E staining or gold when labelled with nile red were detected in the WFB-enriched fraction (Fig. 5A and B). Based on a comparison of morphology, staining characteristics and size with the WFBs observed in isolated macrogametocytes (Fig. 5A, insert), it was concluded that the organelles detected in these fractions are indeed intact parasite Type 1 and 2 WFBs (Fig. 5B, insert). This view was further supported by immunofluorescence analysis which showed that the ‘doughnut-shaped’ WFB2s reacted strongly with the 1E11-11 monoclonal antibody to EmGAM 56 (Fig. 5C), whereas the large WFB1s reacted strongly with the polyclonal anti-APGA and the monoclonal E1D8 antibodies (Fig. 5D and E). The E1D8 antibody reacted exclusively with the membrane surrounding the extracted WFB1s of E. maxima, as shown in the two-dimensional focal slice of the same sample (Fig. 5E, insert). Interestingly, after a period of incubation the WFB1s seen in the detergent suspension spontaneously aggregated into ‘necklace-like’ structures similar to that seen under the surface of purified macrogametocytes (Fig. 5A–E).
Light microscopy and fluorescent micrographs of the WFB-rich extract. (A) Light microscopy of the WFB-rich extract stained with H&E, highlighting extraction of the large WFB1s and their arrangement into necklace-like structures similar to that seen in the macrogametocytes harvested from the chicken intestine 134 h p.i. (insert). (B) The WFB-rich extract (same to that shown in (A)) incubated with nile red showing the localization of the lipids (yellow) to the extracted large WFB1s and glycoproteins and/or polar lipids (orange/red) to the ‘doughnut-shaped’ aggregated WFB2s (insert). (C) Immunofluorescent micrographs of the WFB-rich extract incubated with a monoclonal antibody 1E11-11 specific to EmGAM56, showing the clustered 1E11-11-positive ‘doughnut-shaped’ WFB2s (green). (D) An immunostained image of the WFB-rich extract showing the large APGA-positive WFB1s (green) clustered into ‘necklace-like’ structures. (E) The WFB-rich extract was incubated with a monoclonal anti-WFB1 antibody E1D8 and analysed by confocal laser scanning microscopy. Shown is the three-dimensional confocal reconstruction of immunofluorescence signals. Note the aggregation of the E1D8-positive WFB1s and the E1D8 accumulation (green) to the membrane located at the surface of the WFB1 organelle (insert; the three-dimensional confocal reconstructions of through-focal optical section). Bar = 2 μm.
The lysates of isolated gametocytes and uninfected enterocytes, as well as an extract of isolated WFBs, were analysed by SDS–PAGE. Silver staining of proteins collected from the enterocyte host control lysate showed a number of protein bands migrating between 170 and 15 kDa, with the 170, 135, 35 and 17 kDa bands being the most prominently stained (Fig. 6A, Host). In comparison, the gametocyte lysate showed a very different banding pattern, and several of the major protein bands observed in the enterocyte lysate were undetectable in the silver-stained gametocyte extract, indicating that there was minimal host protein contamination. Of all the parasite-specific proteins detected in the gametocyte lysate, the 62, 58 and 56 kDa bands were the most prominent, representing a significant proportion of the total protein extracted (Fig. 6A, Gametocyte). Four prominent bands were also detected between 80 and 120 kDa and several other prominent bands were observed between 12 and 45 kDa in this lysate. One of these bands of 17 kDa was RNase which was used in the extraction process.
SDS-PAGE and Western blot, in which lane 1 shows the uninfected control enterocytes, lane 2 shows the gametocyte lysate, lane 3 shows the WFB-rich extract (colloid suspension formed by the detergent treatment of the pelleted lysate) retained on top of the 1000 kDa filter. (A) A silver-stained gel of extracts from uninfected enterocytes, gametocytes and WFBs. (B) An immunoblot probed with anti-WFB2 monoclonal antibody, 1E11-11. Note the major gametocyte antigen, EmGAM 54/56 doublet (lanes 2 and 3). (C) An immunoblot probed with anti-APGA. The 3 gels were loaded with 4 μg of protein per lane. The position and molecular mass (in kDa) of protein standards are indicated on the right.
Prominent protein bands having molecular weights of 62, 58 and 56 kDa were also visualized in the extract prepared from isolated WFBs (Fig. 6A). In addition, 4 bands ranging in size between 80 and 120 kDa were also observed in the extracted WFBs. Finally, there were only 2 smaller molecular weight proteins of 45 and 38 kDa observed in the extracted WFBs with the complete absence of the RNase band, which further supports the contention that fractionation has resulted in the isolation of an extract that is substantially enriched with the WFBs.
By Western blotting, using the mouse anti-WFB2 monoclonal antibody 1E11-11, the gametocyte protein EmGAM 56 was detected as a doublet 56/54 kDa band in the gametocyte lysate and the extracted WFBs (Fig. 6B). The only difference was that it appears that the relative amount of the 56 kDa band within the doublet is higher in the extracted WFBs as compared with the starting gametocyte lysate. As a control, the uninfected enterocyte extract probed with 1E11-11 antibody showed no reactivity (Fig. 6B, Host).
Using anti-APGA on a Western blot we found that the 3 major gametocyte protein bands of 56/54, 58 and 62 kDa all reacted with the antiserum (Fig. 6C). Only when we loaded 10-fold more protein were we able to detect the 82 and 230 kDa proteins (not shown). Interestingly, using anti-APGA the 56/54 doublet band showed that the 54 kDa protein lower band was greater in intensity than the upper band in contrast to the opposite result seen using the monoclonal antibody to the 56 kDa antigen.
DISCUSSION
The results of the present study show that isolated gametocytes are both intact and metabolically active. This is based on the finding that the wall forming bodiess in isolated macrogametocytes have the same size, shape and staining properties as those seen in tissue sections. In addition, it was found by time-lapse imaging of purified macrogametocytes that during in vitro incubation the WFBs become visible in the periphery of the parasite with a corresponding increase in autofluorescence. Furthermore, we observed the presence of well-formed microgametes budding off from the microgametocyte. These results, together with a previous study showing the incorporation of L-[S35] methionine into antigenic proteins from isolated gametocytes (Mencher et al. Reference Mencher, Pugatsch and Wallach1989; Pugatsch et al. Reference Pugatsch, Mencher and Wallach1989), indicate that macrogametocyte maturation does continue even after E. maxima gametocytes are removed from the infected intestine. Our results also indicate that the enzymes and other molecules required for parasite growth and development are intact and active in purified gametocytes although further work is required to demonstrate their presence.
During the course of gametocyte development, there is a large burst in the biosynthesis of WFBs in which nearly all of its cytoplasm is filled by WFBs. Nile red and oil red O staining showed that the WFB1s migrate to the periphery of the parasite and release their contents in patches in the empty space between the 2 macrogametocyte-specific membranes. Nile red staining of purified sporulated oocysts indicates that these patches eventually fuse together to form the outer wall of the oocyst. It was also observed that the WFB1s form links and chains on the surface of the parasite indicating the start of WFB1 joining and exocytosis. Shortly afterwards, the WFB2s start to fuse, forming the inner layer of the oocyst wall through proteolytic processing and di-tyrosine cross-linking of EmGAM56 and EmGAM82 as described previously (Belli et al. Reference Belli, Wallach, Luxford, Davies and Smith2003; Mai et al. Reference Mai, Sharman, Walker, Katrib, De Souza, McConville, Wallach, Belli, Ferguson and Smith2009).
In our studies using nile red we found that the WFB1s and outer oocyst wall stained bright yellow gold. This result indicates that the major components of these organelles are neutral lipids such as cholesterol and triglycerides. Previous studies from our laboratory have shown that the oocyst wall contains relatively large quantities of cholesterol and triglycerides even after bleaching of both unsporulated or sporulated oocysts (Mai et al. Reference Mai, Sharman, Walker, Katrib, De Souza, McConville, Wallach, Belli, Ferguson and Smith2009). Therefore, it was not surprising to find that both nile red and oil red O, which are sensitive lipophilic probes, strongly reacted with the WFB1s, lighting up the entire organelle.
It was found that a monoclonal antibody previously shown to specifically bind to the WFB1s of E. tenella (Mouafo et al. Reference Mouafo, Weck-Heimann, Dubremetz and Entzeroth2002) reacted with antigens localized to the membrane surrounding the WFB1s of E. maxima. This was particularly evident in a z-series of confocal images collected through the isolated WFB1s, which showed the concentration of green fluorescence in the rim of the organelle (Fig. 5E). Together with nile red results, it was therefore postulated that these organelles contain a lipid core with a protein-rich surface coat. The role of this WFB1 protein in outer oocyst wall formation is currently being investigated.
A simple method to extract WFBs from purified gametocytes was developed and it was shown by light and fluorescent microscopy, immunofluorescence and Western blotting that they were intact and contained the major WFB1 and WFB2 antigens. In addition, it was shown by SDS-PAGE that in both gametocyte lysate and WFB-enriched fractions, the 56, 58 and 62 kDa antigens were the most prominent bands, representing a large proportion of the total gametocyte protein. Based on silver staining of the SDS-PAGE gel, it appeared that the extraction procedure resulted in a significant enrichment of the major WFB proteins as most of the smaller molecular weight proteins detected in the gametocyte lysate were absent in the WFB-rich extract.
We had shown previously that the 56 and 82 kDa gametocyte antigens are partially insoluble even in strong detergent (Belli et al. 2002a), and based on this study it would appear that these organelles form a stable colloidal suspension in 1% SDS. This conclusion is supported by the finding that even after concentrating and washing the organelles with PBS buffer using a 1000 kDa MWCO filter, the major WFB1 and WFB2 proteins remained associated with the intact WFBs. This biophysical property of the WFBs is most likely due to the relative insolubility of the major protein and lipid components of these organelles, which maintain their integrity until a signal is given to release their contents possibly after fertilization.
The results of the present study help to elucidate the biosynthesis and spatial organization of WFBs during the development of the oocyst wall of E. maxima using isolated and purified gametocytes. The picture that emerges from all of these studies is as follows: as the macrogametocytes mature the veil-forming and WFBs are synthesized as small vesicles that enlarge and spread out in the cytoplasm during development. Once the parasite has reached its final size the veil, which contains mainly proteins/glycoproteins, is laid down surrounding the parasite membrane. In mid- and late-stage macrogametocytes the neutral lipid containing WFB1s align themselves at the periphery of the parasite, form links and chains, and then their contents are exocytosed as lipid patches (‘rafts’) into the space between 2 parasite membranes in a patchwork fashion. During outer wall formation the WFB2s, which are rich in the glycoproteins EmGAM 56, EmGAM 82 and EmGAM 230, begin to coalesce in the gametocyte cytoplasm. Then, these tyrosine-rich glycoproteins are enzymatically cleaved into smaller peptides that are cross-linked and inserted into the inner wall. Finally, there are some ‘leftover’ WFBs that remain in the cytoplasm of the newly formed oocyst/zygote.
Acknowledgements
We are grateful to the following people: the team at the Ernst Facility for maintaining the chickens, Dr Marilyn Katrib for advice on time-lapse imaging, Dr Jan Slapeta (The Faculty of Veterinary Science, University of Sydney, New South Wales) for preparing paraffin sections and Phillipa Sharman (The iThree Institute, University of Technology Sydney) for her assistance in the isolation of parasites. The authors are also thankful to Dr Lisa Sedger (The iThree Institute, University of Technology Sydney) and Dr Jan Slapeta for their critical assessment of the work presented here.
FINANCIAL SUPPORT
The authors also acknowledge the financial support provided through the Australian Research Council Linkage Grant in partnership with PhiBro Animal Health Corporation, Israel.
