Maternal inheritance and stage-specific variation of the apicoplast in Toxoplasma gondii during development in the intermediate and definitive host

Abstract

The structure and location of Toxoplasma gondii apicoplasts were examined in intermediate and definitive hosts and shown to vary in a stage-specific manner. Immunocytochemistry and electron microscopy studies were used to identify changes in the morphology of apicoplasts and in their enoyl reductase (ENR) content during asexual and sexual development. Apicoplasts in tachyzoites were small, multimembraned organelles anterior to nuclei that divided and segregated with the nuclei during endodyogeny. In nonproliferating bradyzoites within mature tissue cysts (1 to 24 months), apicoplasts had high levels of ENR. During coccidian development, asexual multiplication (endopolygeny), resulting in simultaneous formation of up to 30 daughters (merozoites), involved an initial growth phase associated with repeated nuclear divisions during which apicoplasts appeared as single, elongated, branched structures with increased levels of ENR. At initiation of merozoite formation, enlarged apicoplasts divided simultaneously, with constrictions, into portions that segregated to developing daughters. In sexual stages, apicoplast division did not occur during microgametogony, and apicoplasts were absent from the microgametes that were formed. In contrast, during macrogametogony, the apicoplast appeared as a large, branched, perinuclear structure that had very high levels of ENR in the absence of nuclear division. Marked increases in the size of apicoplasts and levels of ENR may be related to requirements of the macrogametocytes to synthesize and store all components necessary for oocyst formation and subsequent extracellular sporulation. Thus, it is shown that apicoplasts are present and contain ENR in all T. gondii life cycle stages except microgametes, which will result in maternal inheritance of the organelle.

FIG. 1.

Western blot of T. gondii recombinant ENR showing the negative result with the preimmune serum while the anti-ENR serum recognizes a 33-kDa protein, the predicted molecular mass of ENR.

FIG. 2.

Immunofluorescent images through the brains of mice infected with T. gondii at 15 days (a to e and g), 21 days (h), and 15 months (f, i, and j) postinfection. Sections were stained with anti-SAG1/anti-BAG1 (a), anti-SAG1/anti-ENR (b to e), or anti-BAG1/anti-ENR (f to i) and visualized with secondary antibodies conjugated to Texas red (red) and FITC (green), respectively, and the nuclei were counterstained with DAPI (blue). Bars, 5 (a, b, and f) and 1 μm (c, d e, g, h, and i). (a) Small lesion in which parasites undergoing stage conversion can be identified by the presence of both SAG1⁺ tachyzoites (T; red) and early cysts (Cy) containing BAG1⁺ bradyzoites (green). (b) Similar lesion to that shown in panel a in which the tachyzoites (T) can be identified as positive for SAG1 (red) while the early tissue cysts (Cy) are unstained. It was observed that theapicoplasts in both the tachyzoites and bradyzoites were positively stained with anti-ENR (green). (c to e) Details of SAG1⁺ tachyzoites (red) showing the apicoplast stained with anti-ENR (green). Note the range in shape from spherical in panel c to an elongated structure in panel d and finally division into two in panel e. (f) A mature tissue cyst (15 months postinfection) showing numerous bradyzoites with BAG1⁺ cytoplasm (red) and ENR⁺ apicoplasts anterior to the nuclei (arrows). (g) An early small tissue cyst (15 days postinfection) with a few BAG1⁺ bradyzoites (red). Note that the bradyzoites possess an elongated ENR⁺ apicoplast (green) just anterior to the nucleus. (h) Small tissue cyst containing BAG1⁺ bradyzoites (red). Note that certain bradyzoites appear to contain possibly two ENR-positive (green) apicoplasts (arrowheads). (i) Detail from panel f showing the presence of enlarged ENR⁺ apicoplasts (green) within the mature bradyzoites. (j) Section stained with hematoxylin and eosin showing the morphology of a tissue cyst in mouse brain. N, nucleus.

FIG. 3.

Ultrastructural appearances of the apicoplast during tachyzoite (a and b) and bradyzoite (c to f) development. (a) Electron micrograph of a section through intracellular tachyzoite showing an enlarged mitochondrion (Mi) and an elongated structure (arrowhead) anterior to the nucleus (N). Bar, 500 nm. (b) Enlargement of the enclosed area shown in panel a showing the nucleus, the Golgi body (G), and the multimembranous apicoplast (arrow). Note that the membranes are less distinct on the face adjacent to the Golgi body (arrowheads). Bar, 100 nm. (c) Detail of the anterior cytoplasm of a bradyzoite illustrating an elongated multimembraned apicoplast (arrows). Bar, 100 nm. (d) Enlargement of a serial section of the upper apicoplast shown in panel f in which four membranes can be readily identified along the lateral surfaces (arrowheads). Bar, 100 nm. (e) Low-power electron micrograph of a section through a bradyzoite showing the posteriorly located nucleus (N), numerous polysaccharide granules (PG), micronemes (M), and rhoptry (R). CW, tissue cyst wall. Bar, 500 nm. (f) Enlargement of the enclosed area shown in panel e showing the Golgi body (G) anterior of which are dense granules (DG) and profiles of two apicoplasts (A). Bar, 100 nm.

FIG. 4.

Sections illustrating the coccidian (asexual and sexual) development of T. gondii in the villus of the small intestine of the cat. Panels a, d, and i show 1-μm plastic sections stained with Azure A from material processed for electron microscopy. Panels b, c, e, f, h, j, k, and l are sections that have been immunolabeled with anti-ENR in combination with other primary antibodies visualized with FITC (green) and Texas red (red), respectively. Panel g shows a control section stained with preimmune sera. The nuclei stained with DAPI (blue). Bars, 5 μm. (a) Part of a villus showing the morphological appearances of the developing parasites within the enterocytes. Trophozites (Tr), macrogametocytes (Ma), and multinucleate parasites can be identified. (b) Low-power longitudinal section of a villus stained for ENR (green) and NTPase (red) showing the parasitophorous vacuoles positive for NTPase (red). Note the small ENR⁺ (green) apicoplasts associated with the small uninuclear parasites (arrowheads) but the larger structure and stronger signal from the multinucleate stage (arrow). (c) Section of a villus stained for ENR (green) and ENO2 (red) showing numerous strongly labeled nuclei for ENO2 of parasites with single and multiple nuclei. Note the strongly labeled large elongated structure centrally located in the multinucleate mid-stage schizonts (arrows) in comparison to the small structure associated with the uninuclear trophozoite (arrowhead). (d) Enlargement of a multinucleate mid-stage schizont. (e) Section stained for ENR (green) and ENO2 (red) showing an elongate apicoplast (arrow) stretching between the ENO⁺ (red) nuclei. Possible constriction in the apicoplast can be seen (arrowheads). (f) A mid-stage schizont single labeled for ENR (green) showing the complex branched structure of the apicoplast (arrow). (g) Control section stained with the preimmune serum showing the absence of any ENR labeling (green). The presence of parasites is confirmed by the DAPI staining of the nuclei. (h) Section stained for ENR (green) and ENO2 (red) showing a number of ENO2 positive nuclei and an apparently fragmented apicoplast (A). (i) Detail of a mature schizont showing the structure of the fully formed merozoites (Me). (j) Section through a mature schizont stained for ENR (green) and NTPase (red) showing the dense granules (DG) in the apical cytoplasm of the merozoites with the small apicoplast (arrowheads) located between them and the nucleus. (k) Extracellular merozoites stained for ENR (green) and ENO2 (red), which in the mature merozoites only stains the cytoplasm, showing the small spherical apicoplast (arrowhead) located adjacent to the nucleus. (l) Extracellular merozoites stained for ENR (green) and NTPase (red) showing the small spherical apicoplast located between the nucleus and the dense granules (DG). N, nucleus.

FIG. 5.

Electron micrographs illustrating the early stages of asexual development in the small intestine of the cat. (a) An early intracellular trophozoite showing the nucleus, Golgi body, mitochondrion, and apicoplast. Bar, 1 μm. (b) Later and larger trophozoite showing the increase in the size of the mitochondria and the apicoplast. Bar, 1 μm. (c) Mid-stage schizont with a number of nuclei and a centrally located elongated apicoplast. Bar, 1 μm. (d and e) Details from a multinucleated schizonts similar to that shown in panel c showing a number of profiles through the multimembraned apicoplast. Bar, 100 nm. (Inset) Enlargement of the enclosed area in panel d in which the presence of four membranes can be resolved (arrowheads). Bar, 100 nm. A, apicoplast; N, nucleus; G, Golgi body; Mi, mitochondria.

FIG. 6.

(a) Low-power electron micrograph of a section through a late-stage schizont in which the early stages in the formation of a number of daughters (D) can be seen within the cytoplasm. Bar, 1 μm. (b) Detail of part of panel a showing the inner membrane complex of the developing daughters becoming associated with the nuclei. Note an apicoplast apparently entering a daughter. C, conoid; R, rhoptry; bar, 100 nm. (c) Detail of the cytoplasm of a late-stage schizont showing the formation of the apical end of the daughters within the cytoplasm. The daughters consist of the conoid (C), rhoptry precursor (R), and the membrane complex directed toward the nuclei. At this time apicoplasts can be seen adjacent to the nuclear pole (NP) and apparently entering the developing daughter. G, Golgi body; bar, 100 nm. (d) Part of a mature schizont showing the fully formed merozoite containing a nucleus, rhoptries (R), dense granules (DG), and small apicoplast adjacent to the nucleus. Bar, 500 nm. N, nucleus; A, apicoplast.

FIG. 7.

Sections illustrating the sexual development of T. gondii in the villi of the small intestine of the cat. Panels a and c show 1-μm plastic sections stained with Azure A from material processed for electron microscopy. Panel b and panels c to j are sections that have been immunolabeled with anti-ENR in combination with other primary antibodies visualized with FITC (green) and Texas red (red), respectively. Bars, 5 μm. (a) Mature microgametocyte with a few microgametes, identified by their dense staining nucleus, located within the parasitophorous vacuole. (b) Similar stage to that shown in panel a immunostained for ENR (green) showing a single apicoplast (arrow) with a relatively low level of signal, while the microgamete nuclei are strongly stained with DAPI. (c) Section through a macrogametocyte showing the large nucleus with its nucleolus and the cytoplasm packed with granules. (d and e) Sections through macrogametes stained for ENR (green) and ENO2 (red) showing the cytoplasm labeled for ENO2 and a very strong ENR signal from a centrally located irregular shaped structure (arrow). (f) Macrogametes stained for ENR (green) and NTPase (red) showing NTPase labeling of the parasitophorous vacuole (PV) and ENR⁺ structure associated with the central nucleus. (g to j) Duplicate images of sections stained for ENR (green) and ENO2 (red) but in which one image shows saturation of the ENR signal within the apicoplast (arrow) of the multinucleate mid-stage schizont (g) or macrogametocyte (i), at which point it is difficult to identify the apicoplasts within the merozoites of the mature schizont (arrowheads). In the second pair of images (h and j) the presence of the apicoplasts within the merozoites was confirmed by increasing the level of signal in the green channel (arrowheads). N, nucleus.

FIG. 8.

(a) Mature macrogametocyte showing the centrally located nucleus (N) with adjacent apicoplasts (A). The cytoplasm contains a number of wall-forming bodies type 1 (W1), a few wall-forming bodies type 2 (W2), and numerous polysaccharide granules (PG) and lipid droplets (L). Bar, 1 μm. (b) Enlargement of the apicoplast shown in panel a illustrating the complex shape and the formation of constriction rings (arrows). Bar, 100 nm. (c) Detail of an apicoplast within a developing schizont showing ring-like constriction (arrow). Bar, 100 nm.