Thin section studies of residual bodies

The total in vitro excystate from C. parvum oocysts contains a mixture of intact oocysts, partially excysted oocysts, free sporozoites, free residual bodies (some damaged) and empty oocyst walls. Figure 1 shows a thin section of a partly excysted oocyst, with one banana-shaped sporozoite and the residual body within the oocyst wall. The membrane-enclosed residual body contains a characteristic large lipid body, numerous amylopectin granules and a crystalline protein inclusion, along with more finely dispersed material (ribosomes) and cytomembranes.

Fig. 1. A thin-sectioned Cryptosporidium parvum oocyst showing the presence of the residual body (Rb) and one of the remaining ‘banana-shaped’ sporozoites (Sp) within the oocyst wall (OW). Within the membrane-bounded residual body, a lipid body (L), protein crystal inclusion (CI) and lightly stained amylopecin granules (A) can be defined, along with internal cytomembranes, vacuoles and particulate material. The sporozoite contains a number of different organelles, particles and granules (e.g. nucleus, micronemes, rhoptry, dense granules, ribosomes, crystalloid body particles (see Petry & Harris, 1999), and possibly also some amylopectin granules (arrowheads) which are significantly smaller than those in the residual body.

Some small particles within the sporozoite are also stained with a similar density to the amylopectin granules in the residual body (indicated by arrowheads in Fig. 1). Whilst amylopectin is often present within the mature and immature apicomplexan parasites, it has not always been convincingly demonstrated within C. parvum sporozoites. As the sporozoite shown in Fig. 1 possesses clearly defined micronemes and dense granules, it is likely that the more lightly stained granules are indeed amylopectin. Figure 2 shows a montage with emphasis upon the residual body remaining within the wall of the excysted oocyst. Variable density of stained amylopectin granules has often been observed; indeed they often tend to appear as electron-lucent particles (Fig. 2A), or as more commonly found in our data, lightly stained but with a more densely stained surface (see below). The crystalline inclusion does not appear in all sections through the residual body, but when it is present it has features characteristic of a protein crystal (Fig. 2B). When the residual body escapes from the oocyst wall it has a tendency to undergo disruption with release of its contents, but many remain intact. Figure 3 shows examples of thin-sectioned externalized residual bodies that are relatively intact. At low magnification (Fig. 3A) the distribution of amylopectin granules, stained relatively intensely in this instance, can be seen. The amylopectin granules have irregular oval shape, but they all exhibit a smooth surface and there is a narrow size range (approx. 0·1–0·3 μm). At higher magnification (Fig. 3B) the amylopectin granules can be seen to possess a fine granular interior which shows some indication of linearity, suggestive of ordered substructure (Fig. 3C).

Fig. 2. Thin sections showing residual bodies still within the Cryptosporidium parvum oocyst wall after excystation. (A) Residual body in the process of exiting through the suture of the oocyst wall (OW). In this instance the surrounding membrane of the residual body is still intact; the amylopectin granules and lipid body (L) are relatively lightly stained. (B) Fragmented residual body exiting the oocyst wall suture, with more densely stained amylopectin granules than in (A) and a crystal inclusion showing parallel dense lines characteristic of a stained protein crystal (arrowheads).

Fig. 3. Thin sections showing Cryptosporidium parvum residual bodies outside the oocyst wall, following excystation. (A) Two residual bodies, each with a relatively intact membrane, well-defined amylopectin granules and lipid body (L). (B) At slightly higher magnification the peripheral densely stained rim of the amylopectin granules is emphasized together with the finely stained nature of the granule content. (C) At high magnification, free amylopectin granules and those within a relatively intact residual body show an internal particulate structure, which occasionally possesses an indication of linearity (view between arrowhead pairs).

Negative staining of intact and disrupting residual bodies

The inherent thickness and density of intact C. parvum residual bodies and sporozoites usually prevents the possibility of obtaining information about their contents from the negative staining technique. If, however, the residual body has undergone partial disruption in suspension and/or at the time of producing the negatively stained specimen, then it is possible to define some of the internal components, although excessive stain density can still be a problem (Fig. 4A). When 1% or 0·5% ammonium molybdate is used for negative staining instead of the more usual 2%, then there is a greater chance that detail will be revealed (Fig. 4B). In the total excystate, the protein crystalline inclusions that have been released from disrupted residual bodies are readily revealed by negative staining (Fig. 4C), thereby providing confirmation of the structural interpretation placed on the thin section data (Figs 1 and 2).

Fig. 4. (A) A more thinly spread negatively stained residual body undergoing disruption, lying alongside an oocyst wall (OW). The numerous electron-dense amylopectin granules surround an electron-transparent irregular lipid body (L). This electron micrograph serves to show the inherent technical difficulty when applying the negative stain procedure to relatively large and thick samples of biological material such as the oocyst and residual body. (B) Negatively stained Cryptosporidium parvum residual body in the total excystate, undergoing disruption. Although rather electron dense, in this instance the negative stain does reveal more clearly than in (A) the amylopectin granules (arrowheads) surrounding the lipid body (L). (C) A cluster of negatively stained crystal inclusions, present in the total excystate, showing linear periodicity characteristic of a protein crystal (cf. Fig. 2B).

Negative staining of isolated amylopectin granules

Amylopectin granules isolated as the sucrose gradient pellet following French press disruption of the sporozoites and residual bodies have been found to show a considerable size range. A survey micrograph of a negatively stained sample (Fig. 5A) shows that the population of larger granules exhibit a smooth surface with a compact tightly rolled internal appearance, resembling a ball of string. The smaller amylopectin granules have a more irregular surface and particulate internal content. These structural features of the two classes of amylopectin granules are emphasized at higher magnification in Fig. 5B–D. Sucrose-gradient pellets obtained directly from the total excystate, which contains many disrupted residual bodies, but intact sporozoites, yielded an amylopectin pellet containing only the larger population of granules (Fig. 5B, C). The neatly coiled, smooth-surfaced ‘ball of string’ structure of the larger amylopectin (Fig. 5B, C) contrasts markedly with the more irregular shape and rod-like particulate composition of the smaller granules (Fig. 5D). The smaller amylopectin granules (Fig. 5D) are thought to be derived from the sporozoites, as sucrose gradient centrifugation performed using a purified sporozoite fraction from the C. parvum excystate produced a pellet containing only the population of smaller irregular granules. They could represent a residual amylopectin core that has been metabolically degraded from initially larger granules within the sporozoites or a unique population of smaller undegraded amylopectin granules within the sporozoite.

Fig. 5. (A) Survey electron micrograph showing the mixed population of large and small negatively stained amylopectin granules on a continuous carbon support film. Samples were obtained by sucrose density-gradient centrifugation, following disruption of Cryptosporidium parvum sporozoites and residual bodies in the total excystate using a French pressure cell (Petry & Harris, 1999). Note the compact, smooth-surfaced appearance of the larger granules. Trapped water within the granules often presents a problem, resulting in visible electron beam damage (arrowheads). At higher magnification the structural details of the 2 populations of granules become more apparent. (B) and (C) Larger granules, purified from residual bodies which had previously been separated from sporozoites, which exhibit a tightly coiled ‘ball of string’ morphology, possibly due to the linear structure of the amylopectin polysaccharide chains as flexible coiled ~5 nm diameter strings. (D) The purified population of smaller amylopectin granules, derived from sporozoites first separated from residual bodies by sucrose-gradient centrifugation. The irregularity of the clusters of rod-like particles composing these small granules contrasts strongly with the purified smooth-surfaced larger amylopectin granules from the residual bodies (B, C).

Apart from conventional negative staining, with the sample adsorbed to a carbon support film, several other negative staining and cryoelectron microscopy approaches have been used to study the structure of the C. parvum amylopectin granules. Figure 6 shows a composite with amylopectin granules negatively stained (Fig. 6A) and unstained (in the presence of trehalose; Fig. 6B) after spreading across the holes of a holey carbon support film and air-drying (Harris & Scheffler, 2002). The corresponding cryoelectron microscopy images are shown of cryo-negatively stained amylopectin (Fig. 6C) and unstained frozen-hydrated amylopectin (Fig. 6D), (Adrian et al. 1984, 1998).

Fig. 6. Amylopectin granules imaged following differing specimen preparation conditions. (A) Negatively stained by spreading in 5% ammonium molybdate, 0·1% trehalose (pH 7·0) across a holey carbon support film and air-dried. (B) Spread across a holey carbon support film in the presence of 1% trehalose and air-dried (i.e. unstained). (C) Cryo-negatively stained with 15% ammonium molybdate (pH 7·0), on a holey carbon support film. (D) Vitrified directly, in the absence of negative stain, on a holey carbon support film (i.e. cryo-electron microscopy). For both the negatively stained examples (A and C) the ‘ball of string’ structure of the oligosaccharide within the amylopectin granules is clearly shown, and with the fully hydrated sample (C) it can be assumed that there is no shrinkage artefact induced by drying. The unstained samples (B and D), show images produced only by the inherent electron density of the amylopectin granules which, in the case of 1% trehalose embedding (B) (perhaps with an element of contrast matching), does not reveal any detail other than the overall size of the particle. However, conventional cryo-electron microscopy (D) does reveal the unstained amylopectin with greater clarity than 1% trehalose and there is a slight indication of the polysaccharide substructure. In general, these alternative technical approaches provide support for the validity of the amylopectin images shown in Fig. 5.

The absence of a carbon support film creates the potential for greater image detail from the negatively stained amylopectin (Fig. 6A). The frozen-hydrated negatively stained images of the amylopectin granules shown in Fig. 6C reveal a more tightly packed internal structure, but in general these images correlate well with those shown above (Figs 5 and 6A). No internal detail within unstained amylopectin is revealed by air drying in a thin film of trehalose (Fig. 6B). Undoubtedly there will be an element of contrast matching between the disaccharide trehalose and amylopectin, which explains the indistinct edge of amylopectin granules. With cryoelectron microscopy, however, unstained vitrified specimens of amylopectin reveal the granule edge more clearly (Fig. 6D), since the density of the surrounding vitreous ice is significantly less than that of the amylopectin. Nevertheless, the internal structure within the amylopectin granules is only just definable under these conditions (Fig. 6D).

Glycohydrolase and heat treatment of amylopectin granules

The intense blue colour of starch in the presence of iodine is a characteristic colouration reaction for the detection of amylose and amylopectin. C. parvum amylopectin granules do indeed produce this same colouration (data not shown) and from our earlier biochemical analysis (Petry & Harris, 1999) we know that there is no protein in the amylopectin pellet taken from our sucrose gradients. Digestion of the amylopectin granules with enzymes known to cleave complex branched polysaccharide chains is likely to yield useful structural information as to the granule composition. We have investigated the action of amyloglucosidase, α-amylase, β-amylase and pullulanase, with incubation at room temperature and at 40 °C. Amylopectin granules subjected to digestion for several hours, up to 24 h at 40 °C showed a marked tendency to aggregate as small clumps (Fig. 7A, B), with close adherence of one surface to the next, in a viscous manner, but without significant breakdown of the granules. If cleavage of some polysaccharide side-chains occurs, this could create an inherent stickiness causing the granules to adhere to one another, and also account for the intense negative staining (Fig. 7B) and water retention by the particles that can result in electron beam damage (visible in Fig. 7A). Digestion for 24 h at 40 °C with α-amylase produced no aggregation of the amylopectin, instead visible damage to the surface of the particle is clearly apparent (Fig. 7C). The amylopectin granules now have an irregular surface and small rod-like polysaccharide strands are released. There is now an apparent similarity between these large digested amylopectin granules and the population of small possibly sporozoite-derived granules shown in Fig. 5C. β-amylase and pullulanase both produced no significant change to the amylopectin granules, following 24 h digestion (data not shown).

Fig. 7. Amylopectin granules following incubation with glycohydrolases. (A and B) Following incubation at 40 °C for 24 h with amyloglucosidase. The granules in the sample became progressively aggregated, with indication of granule fusion and self-adherence, but without significant breakdown of the granules. The partial digestion of only surface polysaccharide with release of sticky oligosaccharides appears to be responsible for this aggregation, which produces conditions where there is considerable electron beam damage and where there is excessive electron density (B). Following incubation with α-amylase at 40 °C or room temperature for 24 h, some breakdown of the amylopectin granules was detected (C). The granules became irregular in shape, with an uneven surface. Some cleavage of the polysaccharide chains within the granules is apparent, with release of short strands of amylopectin from the granules (arrowheads, C).

Amylopectin (starch) granules are known to become progressively hydrated with increasing temperature and ultimately they form a gel. Treatment of the C. parvum amylopectin granules for short periods of time at 100 °C produced a marked change to their structure (data not shown). After 2 min they lost the compact ‘ball of string’ morphology and had a more globular amorphous structure with fine material radiating out from the centre. After 10 min at 100 °C the particles became swollen, with dispersal of the amylopectin as diffuse globules with an irregular surface. The changes were characteristic of those produced by increasing hydration of amylopectin/starch granules.