The microneme protein MIC4, or an MIC4-like protein, is expressed within the macrogamete and associated with oocyst wall formation in Toxoplasma gondii

The expression and localisation of MIC4, or an immuno-cross reacting MIC4-like protein, was examined in the enteric forms of Toxoplasma gondii using immunocytochemistry. In addition to being located within the micronemes of the merozoites, MIC4 or the MIC4-like protein was present within the macrogamete and was associated with the developing oocyst wall. The macrogamete is characterised by two types of structurally distinct wall forming bodies (WFB1 and 2). However, by immuno-electron microscopy, it was possible to identify two populations of dense granules (WFB1) which appear to form sequentially during macrogamete development. The first granules to form (WFB1a) stained positively with anti-MIC4 and were followed by MIC4 negative granules (WFB1b). During oocyst wall formation, the WFB1a and b sequentially released their contents onto the surface with WFB1a material forming an anti-MIC4 positive outer veil, while the WFB1b forms the electron dense outer layer of the oocyst wall. The inner layer was formed by WFB2. Thus, for the first time, it was possible to identify two populations of dense granules (WFB1a and b) involved in the formation of different parts of the oocyst wall. It was not possible to analyse the contents of macrogametes by western blot to unequivocally identify the antigen recognised by the polyclonal antisera as MIC4.     

Conservation of proteins involved in oocyst wall formation in Eimeria maxima, Eimeria tenella and Eimeria acervulina

Vaccination with proteins from gametocytes of Eimeria maxima protects chickens, via transfer of maternal antibodies, against infection with several species of Eimeria. Antibodies to E. maxima gametocyte proteins recognise proteins in the wall forming bodies of macrogametocytes and oocyst walls of E. maxima, Eimeria tenella and Eimeria acervulina. Homologous genes for two major gametocyte proteins - GAM56 and GAM82 - were found in E. maxima, E. tenella and E. acervulina. Alignment of the predicted protein sequences of these genes reveals that, as well as sharing regions of tyrosine richness, strong homology exists in their amino-terminal regions, where protective antibodies bind. This study confirms the conservation of the roles of GAM56 and GAM82 in oocyst wall formation and shows that antibodies to gametocyte antigens of E. maxima cross-react with homologous proteins in other species, helping to explain cross-species maternal immunity.     

Roles of tyrosine-rich precursor glycoproteins and dityrosine- and 3,4-dihydroxyphenylalanine-mediated protein cross-linking in development of the oocyst wall in the coccidian parasite Eimeria maxima

The oocyst wall of apicomplexan parasites protects them from the harsh external environment, preserving their survival prior to transmission to the next host. If oocyst wall formation could be disrupted, then logically, the cycle of disease transmission could be stopped, and strategies to control infection by several organisms of medical and veterinary importance such as Eimeria, Plasmodium, Toxoplasma, Cyclospora, and Neospora could be developed. Here, we show that two tyrosine-rich precursor glycoproteins, gam56 and gam82, found in specialized organelles (wall-forming bodies) in the sexual stage (macrogamete) of Eimeria maxima are proteolytically processed into smaller glycoproteins, which are then incorporated into the developing oocyst wall. The identification of high concentrations of dityrosine and 3,4-dihydroxyphenylalanine (DOPA) in oocyst extracts by high-pressure liquid chromatography, together with the detection of a UV autofluorescence in intact oocysts, implicates dityrosine- and possibly DOPA-protein cross-links in oocyst wall hardening. In addition, the identification of peroxidase activity in the wall-forming bodies of macrogametes supports the hypothesis that dityrosine- and DOPA-mediated cross-linking might be an enzyme-catalyzed event. As such, the mechanism of oocyst wall formation in Eimeria, is analogous to the underlying mechanisms involved in the stabilization of extracellular matrices in a number of organisms, widely distributed in nature, including insect resilin, nematode cuticles, yeast cell walls, mussel byssal threads, and sea urchin fertilization membranes.     

Fine Structure of Zygotes and Oocysts of Eimeria nieschulzi*

SYNOPSIS. Mature macrogamonts were present in the small intestine of rats 5.5 to 7.5 days postinoculation with Eimeria nieschulzi oocysts; oocysts were present at 6 to 7.5 days. Types I and II wall-forming bodies in macrogamonts began to undergo ultrastructural changes within zygotes to form the outer and inner layers of the oocyst wall. Before and during oocyst wall formation a total of 5 membranes (M1–5) were formed at or near the surface of the zygote. The outer and inner oocyst wall layers formed between M2 and M3, and M4 and M5, respectively. The mature oocyst was loosely surrounded by M1 and M2, had an electron-dense outer layer, 100–275 nm thick, and an electron-lucent inner layer, 160–180 nm thick. It also contained an electron-lucent line consisting of M3 and M4 interposed between the outer and inner layers of the oocyst wall. The micropyle, measuring 935 × 47 nm, was located in the outer layer of the oocyst wall and consisted of 10–14 alternating layers of electron-dense and lucent material. The sporont of mature oocysts was covered by M5, immediately beneath which were M6 and M7. The sporont contained a nucleus and nucleolus, lipid and amylopectin bodies, mitochondria, ribosomes, as well as smooth and rough endoplasmic reticulum. Canaliculi, Golgi complexes, and types I and II wall-forming bodies were absent.     

Molecular characterisation of a novel family of cysteine-rich proteins of Toxoplasma gondii and ultrastructural evidence of oocyst wall localisation

Among apicomplexan parasites, the coccidia and Cryptosporidium spp. are important pathogens of livestock and humans, and the environmentally resistant stage (oocyst) is essential for their transmission. Little is known of the chemical and molecular composition of the oocyst wall. Currently, the only parasite molecules shown to be involved in oocyst wall formation are the tyrosine-rich proteins gam56, gam82 and gam230 of Eimeria spp. and the cysteine-rich proteins COWP1 and COWP8 of Cryptosporidium parvum. In the present study, we searched the ToxoDB database for the presence of putative Toxoplasma gondii oocyst wall proteins (OWPs) and identified seven candidates, herein named TgOWP1 through TgOWP7, showing homology to the Cryptosporidium COWPs. We analysed a cDNA library from partially sporulated oocysts of T. gondii and cloned the full-length cDNAs encoding TgOWP1, TgOWP2 and TgOWP3, which consist of 499, 462 and 640 amino acids, respectively. The three proteins share 24% sequence identity with each other and a markedly similar overall structure, based on the presence of an N-terminal leader peptide followed by tandem duplications of a six-cysteine amino acid motif closely related to the Type I repeat of COWPs. Using antisera to recombinant TgOWP1, TgOWP2 and TgOWP3, we showed by Western blot that these molecules are expressed in T. gondii oocysts but are not detectable in tachyzoites. The solubilisation of TgOWP1–3 strictly depended on the presence of reducing agents, consistent with a likely involvement of these proteins in multimeric complexes mediated by disulphide bridges. Immunofluorescence analysis allowed the localisation of TgOWP1, TgOWP2 and TgOWP3 to the oocyst wall. Additionally, using immunoelectron microscopy and the 1G12 monoclonal antibody, TgOWP3 was specifically detected in the outer layer of the oocyst wall, thus representing the first validated molecular marker of this structure in T. gondii.     

The ultrastructural development of the macrogamete ofEimeria stiedai

Summary The development of the macrogamete ofEimeria. stiedai in the epithelial cells of the bile ducts of rabbits was studied by electron microscopy. A macrogamete was first identified by the presence of a large central nucleus with prominent nucleolus, and subsequently by the appearance of wall forming bodies. The macrogamete was limited by an outer single membrane under which there were remnants of a second membrane. The parasitophorous vacuole, in which the macrogamete was located, was often narrow and it contained no intravacuolar-tubules or -folds. As macrogametogony proceeded wall forming bodies of Type I and II, canaliculi, electron pale spaces (lipid) and polysaccharide granules increased in number. Granular endoplasmic reticulum, mitochondria and Golgi bodies were present throughout.     

Eimeria tenella: further studies on the development of the oocyst

AIM: The present study investigated the processes of macrogametogenesis and oocyst formation of Eimeria tenella (Xiamen strain), including the formation of wall-forming body1 (WFB1) and wall-forming body 2 (WFB2), the club-shape body and the origin of the residual body during the transformation from a macrogamete to an oocyst. METHOD: Transmission electron microscopy was used to follow ultrastructural changes of the organelles during parasite development. Frozen section techniques and special staining were used to determine the chemical composition of the club-shape body. RESULTS: Electron lighter WFB1 appeared earlier than the electron denser WFB2 during the process of cyst wall formation. WFB2 appeared to play a key role in cyst wall formation, whereas WFB1 may have a limited role in the wall-forming process. When two last generation merozoites entered the same host cell simultaneously, one of them grew well, but the other one was developmentally retarded, and became a residual body. Our study indicates that the content of the club-shape body are lipoidal in nature, not amyolpectin as suggested previously, because they stained black by Sudan black-B. CONCLUSIONS: During of macrogametogenesis and oocyst formation of E. tenella (Xiamen strain), WFB2 plays a major role in cyst wall formation. The residual bodies come from the undeveloped macrogametes. The club-body is lipoid; and lipometabolism is important energy resource in E. tenella development.     

Scanning and transmission electron microscopy of the oocyst wall of Isospora lacazei

The oocyst wall of Isospora lacazei from sparrows was studied with scanning (SEM) and transmission (TEM) electron microscopy. In TEM, the oocyst wall consisted of four distinct layers (L1-4). The innermost layer, L1, was moderately electron-lucent and 240--285 nm thick; L2 was electron-dense and 210--240 nm thick; L3 was moderately electron-lucent and 15--150 nm thick; L4, the outer most layer, was discontinuous and consisted of electron-dense discoid bodies which measured 180--220 nm x 320--840 nm. The discoid bodies of L4 as seen by TEM appeared spheroid in shape when observed by SEM. One or two membranes were situated on or between various layers of the oocyst wall. One such membrane occurred on the inner margin of L1, two closely applied membranes were interposed between L1 and L2, one membrane occurred between L2 and L3, and one membrane on the outer margin of L3.     

Ultrastructural studies of the zygote and oocyst wall formation of Eimeria truncata of the lesser snow goose

Zygote development and oocyst wall formation of Eimeria truncata occurred in epithelial cells in renal tubules and ducts of experimentally infected lesser snow geese (Anser c. caerulescens). Post-fertilization stages were present throughout the kidneys beginning nine days post-inoculation. Initially, a single plasmalemma enclosed the zygote, and type 1 wall-forming bodies (WF1) became labyrinthine and moved toward the surface. There, WF1 degranulated and formed the outer layer of the oocyst wall between the plasmalemma and a newly formed second subpellicular membrane. Several WF2 fused and formed the inner layer of the oocyst wall between the third and fourth subpellicular membranes. Six subpellicular membranes were observed during wall formation. Other features of oocyst development were similar to those of other eimerian species.     

Ultrastructural Studies of the Zygote and Oocyst Wall Formation of Eimeria truncata of the Lesser Snow Goose1

ABSTRACT. Zygote development and oocyst wall formation of Eimeria truncata occurred in epithelial cells in renal tubules and ducts of experimentally infected lesser snow geese (Anser c. caerulescens). Post-fertilization stages were present throughout the kidneys beginning nine days post-inoculation. Initially, a single plasmalemma enclosed the zygote, and type 1 wall-forming bodies (WF1) became labyrinthine and moved toward the surface. There, WF1 degranulated and formed the outer layer of the oocyst wall between the plasmalemma and a newly formed second subpellicular membrane. Several WF2 fused and formed the inner layer, of the oocyst wall between the third and fourth subpellicular membranes. Six subpellicular membranes were observed during wall formation. Other features of oocyst development were similar to those of other eimerian species.     

The fine structure of the endogenous stages of Eimeria labbeana: 2. Mature macrogamonts and young oocysts.

The fine structure of the mature macrogamonts and intracellular oocysts of Eimeria labbeana from the ileal mucosa of experimentally infected Pigeons (Columbia livia) was investigated and described. The macrogamont reached a maximum size of 12.0 x 9.5 mum (average equals 10.8 x 8.8 mum), and was located within a narrow parasitophorus vacuole. Most of the macrogamonts were limited by two membranes. Intravacuolar tubules, 1.2 mum long and 58 nm in diameter, established direct connections between the parasite and the host cell. Each tabule was composed of 9 subunits arranged around the central lumen. Cytoplasmic canaliculi were composed of bundles of microtubule-like structures (8-10 nm wide). Type 1 wall-forming bodies reached a maximum size of 1.8 x 1.5 mum, and many had centric or eccentric electron transparent portions within them. They were frequently seen lodged within peripherally-located mitochondria. Type 2 wall-forming bodies averaged 1.5 mum in diameter. The role of the two types of wall-forming bodies in forming the outer and inner layers of the wall of the oocyst was similar to that in other species of Eimeria. The oocyst wall was 0.2 mum thick and composed of a limiting membrane (20 nm thick), an outer layer (75 nm thick), and an inner layer (100 nm thick).     

Behavior of vacuoles during microspore and pollen development in Arabidopsis thaliana

Using the cryo-fixation/freeze-substitution method, we studied the ultrastructural changes and behavior of vacuoles and related organelles (rER and Golgi bodies) during microspore and pollen development, and pollen maturation of Arabidopsis thaliana. In young microspores forming exine (pollen outer cell wall), vacuoles looked like those of somatic cells. In microspores during the formation of intine (inner cell wall), a large vacuole appeared which was made by fusion of pre-existing vacuoles and probably absorption of solutions. In the young pollen grain after the first mitosis, a large vacuole was divided into small vacuoles. The manner of division was not by binary fission and centripetally, but by the invagination of tonoplasts from one side to the opposite side of a vacuole. After the second mitosis, somatic type vacuoles disappeared. In mature pollen grains just before germination, membrane-bound structures containing fine fibrillar substances (MBFs) appeared. The MBFs were considered to be storage vacuoles. In pollen grains from flowers in bloom, MBFs changed to lysosomal structures with acid phosphatases (lytic vacuole). They gradually increased in number and volume, and decomposed the cytoplasm. The autolysis of pollen grains is the first finding in this study, which may contribute to the loss of ability of pollen germination after anthesis.     

Fine structure of the oocyst walls of Isospora serini and Isospora canaria and excystation of Isospora serini from the canary, Serinus canarius L.

Oocysts of Isospora serini and Isospora canaria, from the canary Serinus canarius, were broken, added to a cell suspension, fixed in Karnovsky's fluid, and studied in the electron microscope. The oocyst wall of each species had an electron-lucent inner layer, a more osmiophilic middle layer and an outer layer of electron-lucent (I. serini) or electron-dense material interspersed with some electron-lucent material (I. canaria). A few, relatively large lipid-like bodies were present in the outer or middle layer of the oocyst wall of I. canaria. As many as 9 membranes were present in the oocyst wall of I. canaria and 3 in that of I. serini. When exposed to a trypsin-sodium taurocholate fluid, sporozoites of I. serini excysted from 5-month-old sporocysts in vitro, but not from sporocysts stored for more than 6 months. No excystation occurred in 15-month-old I. canaria sporocysts. Similarities and differences in excystation between I. serini and other Isospora, Eimeria, and Sarcocystis species are discussed.     

Characterization of a developmentally regulated oocyst protein from Eimeria tenella

Changes in proteins during sporulation of Eimeria tenella oocysts were investigated. Unsporulated E. tenella oocysts collected from cecal tissue at 7 days postinoculation were sporulated in aerated media at 28 C for 0-48 hr. Gel analysis of soluble protein extracts prepared from oocysts from their respective time points indicated the presence of 2 prominent bands with relative molecular weight (Mr) in the range of 30 kDa and making up 20% of the total protein. These 2 bands, designated as major oocyst proteins (MOPs), were absent or barely detectable by 21 hr of sporulation. MOP bands were weakly reactive with glycoprotein stain but showed no mobility shift on deglycosylation. By gel analysis it was shown that the purified MOPs consisted of 2 bands of Mr 28.7 and 30.1 kDa. However, by matrix-assisted laser deabsorption-time of flight analysis it was shown that masses were about 17% lower. Internal sequence analysis of the 28.7-kDa protein generated 2 peptides of 17 and 14 amino acids in length, consistent with a recently described protein coded by the gam56 gene and expressed in E. maxima gametocytes. Rabbit antibodies made against MOPs were localized to outer portions of sporocysts before excystment and to the apical end of in vitro-derived sporozoites. These same antibodies were found to react with bands of Mr 101 and 65 kDa by Western blot but did not recognize MOPs in soluble or insoluble sporozoite extracts. The data suggest that the MOPs are derived from part of a gametocyte protein similar to that coded by gam56 and are processed during sporulation into sporocyst and sporozoite proteins. Alternatively, the binding of anti-MOP to 101- and 65-kDa proteins may result from alternatively spliced genes as the development of parasite proceeds.     

Biochemical characterisation of the 56 and 82 kDa immunodominant gametocyte antigens from Eimeria maxima

Two immunodominant gametocyte antigens from Eimeria maxima with M(r) 56 kDa and M(r) 82 kDa have been identified previously as potential candidates for inclusion in a recombinant subunit vaccine against coccidiosis in poultry. Here, these proteins have been biochemically characterised, immunolocalised within the parasite, and sequences for their amino termini determined. These antigens co-purify by affinity chromatography suggesting an interaction with each other. However, separation of the proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of beta-mercaptoethanol did not reveal the presence of inter-chain disulphide bonds. The true masses of the 56 and 82 kDa antigens are 52450 and 62450 Da, respectively, as determined by mass spectrometry. TX-114 separations suggested that they exist, in part, as soluble proteins within the parasite, and immunolocalisation studies indicated that they were found in the wall forming bodies of macrogametocytes. Separation of the proteins by 2D SDS-PAGE revealed that they are acidic in nature and heterogeneous in charge. Cleavage by neuraminidase and O-glycosidase indicated that the presence of O-linked glycans contributed to some of the charge microheterogeneity of both proteins. The absence of these O-glycans however, did not abolish antibody recognition, suggesting that the development of a recombinant subunit vaccine is possible. A more extensive investigation of the carbohydrate moieties of these proteins revealed that they also possess glucose, fucose, mannose and galactose. There was no evidence for the presence of N-linked glycans. The 56 and 82 kDa antigens were separated from a mixture of proteins in a crude gametocyte lysate by 2D SDS-PAGE, the proteins isolated, and the N-terminus amino acid sequence determined. They showed no homology to each other at the N-terminus, or to any other previously characterised protein. Characterisation of these novel proteins has provided further insights into the molecular mechanisms of gametocyte differentiation in E. maxima.     

Assembly and export of a Toxoplasma microneme complex in Giardia lamblia

The microneme proteins of Toxoplasma gondii belong to a large family of adhesins of apicomplexan parasites involved in motility and host cell invasion. During secretory transport, soluble micronemes associate with membrane-bound carriers/escorters and become exposed on the parasite surface as complexes with an array of adhesive domains. Previously, we have exploited the intestinal protozoan Giardia lamblia as an expression system to produce correctly folded and unglycosylated monomeric surface proteins of T. gondii. Here, we report assembly and export of a trimeric microneme (MIC1/4/6) adhesin complex from Toxoplasma. Co-expressed, recombinant microneme proteins were used to investigate structural requirements for microneme complex formation. In addition, export of a microneme subunit induced development of novel Golgi-like compartments demonstrating the existence of post endoplasmic reticulum structures involved in constitutive secretion in this 'Golgi-less' cell. Recreation of the trimeric microneme escorter-cargo system in Giardia is a versatile tool to analyse universal requirements for complex assembly, receptor-ligand interactions and Golgi neogenesis in the basal Giardia secretory system.     

Fine Structure of the Oocyst Walls of Isospora serini and Isospora canaria and Excystation of Isospora serini From the Canary, Serinus canarius L.

SYNOPSIS. Oocysts of Isospora serini and Isospora canaria , from the canary Serinus canarius , were broken, added to a cell suspension, fixed in Karnovsky's fluid, and studied in the electron microscope. The oocyst wall of each species had an electronlucent inner layer, a more osmiophilic middle layer and an outer layer of electron-lucent ( I. serini ) or electron-dense material interspersed with some electron-lucent material ( I. canaria ). A few, relatively large lipid-like bodies were present in the outer or middle layer of the oocyst wall of I. canaria. As many as 9 membranes were present in the oocyst wall of I. canaria and 3 in that of I. serini. When exposed to a trypsin-sodium taurocholate fluid, sporozoites of I. serini excysted from 5-month-old sporocysts in vitro , but not from sporocysts stored for more than 6 months. No excystation occurred in 15-month-old I. canaria sporocysts. Similarities and differences in excystation between I. serini and other Isospora, Eimeria , and Sarcocystis species are discussed.