Correlative light and electron microscopy of wall formation in Eimeria nieschulzi.

Coccidian parasites possess complex life cycles involving asexual proliferation followed by sexual development leading to the production of oocysts. Coccidian oocysts are persistent stages which are secreted by the feces and transmitted from host to host guaranteeing life cycle progression and disease transmission. The robust bilayered oocyst wall is formed from the contents of two organelles, the wall-forming bodies type I and II (WFBI, WFBII), located exclusively in the macrogametocyte. Eimeria nieschulzi has been used as a model parasite to study and follow gametocyte and oocyst development. In this study, the gametocyte and oocyst wall formation of E. nieschulzi was analyzed by electron microscopy and immuno-histology. A monoclonal antibody raised against the macrogametocytes of E. nieschulzi identified a tyrosine-rich glycoprotein (EnGAM82) located in WFBII. Correlative light and electron microscopy was used to examine the vesicle-specific localization and spatial distribution of GAM82-proteins during macrogametocyte maturation by this monoclonal antibody. In early and mid-stages, the GAM82-protein is ubiquitously distributed in WFBII. Few hours later, the protein is arranged in subvesicular structures. It was possible to show that the substructure of WFBII and the spatial distribution of GAM82-proteins probably represent pre-synthesized cross-linked materials prior to the inner oocyst wall formation. Dityrosine-cross-linked gametocyte proteins can also be confirmed and visualized by fluorescence microscopy (UV light, autofluorescence of WFBII).

The chimerical and multifaceted marine acoel Symsagittifera roscoffensis: from photosymbiosis to brain regeneration.

A remarkable example of biological engineering is the capability of some marine animals to take advantage of photosynthesis by hosting symbiotic algae. This capacity, referred to as photosymbiosis, is based on structural and functional complexes that involve two distantly unrelated organisms. These stable photosymbiotic associations between metazoans and photosynthetic protists play fundamental roles in marine ecology as exemplified by reef communities and their vulnerability to global changes threats. Here we introduce a photosymbiotic tidal acoel flatworm, Symsagittifera roscoffensis, and its obligatory green algal photosymbiont, Tetraselmis convolutae (Lack of the algal partner invariably results in acoel lethality emphasizing the mandatory nature of the photosymbiotic algae for the animal's survival). Together they form a composite photosymbiotic unit, which can be reared in controlled conditions that provide easy access to key life-cycle events ranging from early embryogenesis through the induction of photosymbiosis in aposymbiotic juveniles to the emergence of a functional "solar-powered" mature stage. Since it is possible to grow both algae and host under precisely controlled culture conditions, it is now possible to design a range of new experimental protocols that address the mechanisms and evolution of photosymbiosis. S. roscoffensis thus represents an emerging model system with experimental advantages that complement those of other photosymbiotic species, in particular corals. The basal taxonomic position of S. roscoffensis (and acoels in general) also makes it a relevant model for evolutionary studies of development, stem cell biology and regeneration. Finally, it's autotrophic lifestyle and lack of calcification make S. roscoffensis a favorable system to study the role of symbiosis in the response of marine organisms to climate change (e.g., ocean warming and acidification). In this article we summarize the state of knowledge of the biology of S. roscoffensis and its algal partner from studies dating back over a century, and provide an overview of ongoing research efforts that take advantage of this unique system.

Development of Eimeria nieschulzi (Coccidia, Apicomplexa) Gamonts and Oocysts in Primary Fetal Rat Cells.

The in vitro production of gametocytes and oocysts of the apicomplexan parasite genus Eimeria is still a challenge in coccidiosis research. Until today, an in vitro development of gametocytes or oocysts had only been shown in some Eimeria species. For several mammalian Eimeria species, partial developments could be achieved in different cell types, but a development up to gametocytes or oocysts is still lacking. This study compares several permanent cell lines with primary fetal cells of the black rat (Rattus norvegicus) concerning the qualitative in vitro development of the rat parasite Eimeria nieschulzi. With the help of transgenic parasites, the developmental progress was documented. The selected Eimeria nieschulzi strain constitutively expresses the yellow fluorescent protein and a macrogamont specific upregulated red tandem dimer tomato. In the majority of all investigated host cells the development stopped at the second merozoite stage. In a mixed culture of cells derived from inner fetal organs the development of schizont generations I-IV, macrogamonts, and oocysts were observed in crypt-like organoid structures. Microgamonts and microgametes could not be observed and oocysts did not sporulate under air supply. By immunohistology, we could confirm that wild-type E. nieschulzi stages can be found in the crypts of the small intestine. The results of this study may be helpful for characterization of native host cells and for development of an in vitro cultivation system for Eimeria species.

The spatial organization and extraction of the wall-forming bodies of Eimeria maxima.

Eimeria maxima has been used as a model apicomplexan parasite to study sexual stage development and oocyst wall formation. A complete understanding of the wall's biochemical and biophysical properties is of great interest in research on all apicomplexan parasites. Purified gametocytes, zygotes and oocysts were analysed by three-dimensional confocal microscopy, and wide-field fluorescent microscopy was used to investigate the appearance and spatial organization of the 2 types of wall-forming bodies (WFBs). In addition, a variety of staining procedures and immunoassays were used to assess the biosynthesis, metabolic activity, intactness and molecular composition of the WFBs in situ. WFBs were extracted from gametocytes/zygotes and their composition was assessed by microscopy and SDS-PAGE analysis. It was concluded that isolated gametocytes are intact and metabolically active. Additionally, it was observed that the Type 1 WFBs are aligned at the periphery of the parasite and fuse together producing neutral lipid rich patches that appear to be inserted into the space between 2 parasite-specific membranes. Finally, it was shown that the WFBs extracted from purified gametocytes had the same shape, size and staining properties as those observed in situ, and contained the major glycoprotein antigens known to be present in these organelles.

Insight into the ultrastructural organisation of sporulated oocysts of Eimeria nieschulzi (Coccidia, Apicomplexa).

Sporulated oocysts of Eimeria contain four sporocysts with two sporozoites each and a sporocyst residuum. The developing sporozoites are protected by the sporocyst wall and the robust double-layered oocyst wall. Because of problems with conventional fixatives, high-pressure freezing, followed by freeze substitution was used to achieve optimal ultrastructural preservation of oocysts, sporocysts and sporozoites. After embedding in Epon®, ultrathin sections were examined by electron microscopy to select specific oocyst regions for further investigation by electron tomography (ET). ET allows high-resolution three-dimensional views of subcellular structures within the oocysts and sporocysts. Analysis of several 300 nm sections by ET revealed a network of small tubular structures with a diameter of 70-120 nm inside the sporocysts which is decribed here for the first time. This network connects the residual body in a sporocyst with the endoplasmic reticulum (ER) of the surrounding sporozoites. The network consists of membrane-bound tubules that contain vesicles but no larger organelles like mitochondria. These tubules, named "sporocord", may have a function similar to an "umbilical cord" providing the sporozoites with metabolites for long-term survival. Small vesicular structures inside the ER of the sporozoites, multivesicular structures inside the residual bodies and vesicles in the tubules support this hypothesis.

Chimeric fluorescent reporter as a tool for generation of transgenic Eimeria (Apicomplexa, Coccidia) strains with stage specific reporter gene expression.

Progress in transfection of Eimeria sporozoites leads to transformed oocysts, however the output of mutants after passages in the host animals is low. Further enrichment of transgenic oocysts was dependent on fluorescent activated cell sorting and could not be achieved by drug selection. In this study, we fused the Toxoplasma gondii DHFR-TSm2m3 pyrimethamine resistance gene with the yellow fluorescent protein (YFP) encoding sequence to provide continuous pyrimethamine resistance and fluorescence in the Eimeria parasite from a single transcript. The permanent YFP signal of transgenic parasites allows differentiating transgenic parasites from wild type parasites throughout the entire life cycle. The output of transformed oocysts increased up to more than 30% after initial transfection and completion of the life cycle in the host animal. Within three passages under pyrimethamine treatment, a strain with 100% transformed sporulated oocysts of the parasite could be isolated. This new method provides the potential to produce and monitor transgenic Eimeria strains without additional fluorescence activated cell sorting (FACS). The chimeric fluorescent reporter can be utilized as a continuous internal control for plasmids containing stage specific promoter. By this means we utilized an Eimeria tenella gamogony gene specific regulatory sequence to confer macrogamont specific tandem dimer tomato (tdtomato) reporter gene expression in Eimeria nieschulzi.

Improvement of ultrastructural preservation of Eimeria oocysts by microwave-assisted chemical fixation or by high pressure freezing and freeze substitution.

Fixation and preparation for electron microscopy of coccidian oocysts is a general problem. Especially in sporulated oocysts, proper fixation and resin infiltration are hindered by the robust oocyst wall. Conventional chemical fixation therefore leads to artefacts that obscure cellular details in the oocysts. In this study, sporulated oocysts of Eimeria nieschulzi were subjected to different fixation and embedding protocols: conventional chemical fixation and embedding in Spurr's resin, microwave-assisted fixation and processing followed by embedding in epon, and high pressure freezing followed by freeze substitution and epon embedding. The samples were finally studied by transmission electron microscopy. Many ultrastructural features of the oocyst wall and the sporozoites were already substantially improved after microwaved-assisted fixation and processing. However, the fine structural preservation still suffered from shrinkage and artificial extraction, which occured during dehydration and infiltration. High pressure freezing (HPF) and freeze substitution (FS) revealed much better preservation. Oocyst walls retained their ovoid shape, and the ultrastructure of sporozoites was well preserved with no signs of shrinkage or extraction. HPF and FS are therefore a suitable method for the ultrastructural analysis of coccidian oocysts.

Comparison of protective immune responses to apicomplexan parasites.

Members of the phylum Apicomplexa, which includes the species Plasmodium, Eimeria, Toxoplasma, and Babesia amongst others, are the most successful intracellular pathogens known to humankind. The widespread acquisition of antimicrobial resistance to most drugs used to date has sparked a great deal of research and commercial interest in the development of vaccines as alternative control strategies. A few antigens from the asexual and sexual stages of apicomplexan development have been identified and their genes characterised; however, the fine cellular and molecular details of the effector mechanisms crucial for parasite inhibition and stimulation of protective immunity are still not entirely understood. This paper provides an overview of what is currently known about the protective immune response against the various types of apicomplexan parasites and focuses mainly on the similarities of these pathogens and their host interaction. Finally, the evolutionary relationships of these parasites and their hosts, as well as the modulation of immune functions that are critical in determining the outcome of the infection by these pathogenic organisms, are discussed.

New insights into the excystation process and oocyst morphology of rodent Eimeria species.

In this study, the mechanism of excystation of the rodent parasites Eimeria nieschulzi, from rats, and Eimeria falciformis, from mice, was investigated. In vitro, oocysts of both species are susceptible to the protease pepsin, and sporocysts and sporozoites can be excysted in a similar way. Scanning electron microscopy (SEM) revealed a collapse of the oocysts wall at both polar ends after pepsin treatment. This occurs without any visible damage of the outer wall. Using fluorescence and transmission electron microscopy (TEM) we observed that pepsin enters sporulated oocysts at both polar ends and causes degradation of the inner oocyst wall. Using scanning electron microscopy we could identify two polar caps in both investigated rodent Eimeria species, but only one is harbouring the micropyle. Thus the polar caps are the entry site for the pepsin. Furthermore, we provide evidence that the oocyst cap and micropyle are functionally different structures. This study complements the morphological description of both Eimeria species and is of relevance for other coccidian species.

Membrane contact sites between apicoplast and ER in Toxoplasma gondii revealed by electron tomography.

Toxoplasma gondii is an obligate intracellular parasite from the phylum Apicomplexa. A hallmark of these protozoans is the presence of a unique apical complex of organelles that includes the apicoplast, a plastid acquired by secondary endosymbiosis. The apicoplast is indispensible for parasite viability. It harbours a fatty acid biosynthesis type II (FAS II) pathway and plays a key role in the parasite lipid metabolism. Possibly, the apicoplast provides components for the establishment and the maturation of the parasitophorous vacuole, ensuring the successful infection of the host cell. This implies the presence of a transport mechanism for fast and accurate allocation of lipids between the apicoplast and other membrane-bound compartments in the parasite cell. Using a combination of high-pressure freezing, freeze-substitution and electron tomography, we analysed the ultrastructural organization of the apicoplast of T. gondii in relation with the endoplasmic reticulum (ER). This allowed us to clearly show the presence of four continuous membranes surrounding the apicoplast. We present, for the first time, the existence of membrane contact sites between the apicoplast outermost membrane and the ER. We describe the morphological characteristics of these structures and discuss their potential significance for the subcellular distribution of lipids in the parasite.

Reporter gene expression in cell culture stages and oocysts of Eimeria nieschulzi (Coccidia, Apicomplexa).

The rat parasite Eimeria nieschulzi is a suitable model for transfection studies and was used as an additional model organism for the genus Eimeria. We describe the transfection of this apicomplexan parasites and the cultivation of transformed stages in cell culture and in vivo. The beta-galactosidase or yellow fluorescent protein was expressed in all parasitic stages up to the second merozoite generation in vitro under control of the heterologous promoter region of Eimeria tenella mic1 gene previously described for E. tenella transfection. Pyrimethamine resistant E. nieschulzi parasites were obtained in vitro after transfection with a plasmid encoding the Toxoplasma gondii dhfr/ts-m2m3 gene. Co-transfection experiments with an YFP-plasmid resulted in pyrimethamine resistant and fluorescent parasitic stages. Infection of rats with transfected E. nieschulzi sporozoites directed to expression of beta-galactosidase or YFP in oocysts. Co-transfection with YFP/DHFR-TS allowed selection of resistant parasites in vivo. Excreted transgenic oocysts showed arrangement of YFP expression which lead to questions about meiotic recombination frequency and mechanisms.

Improved excystation protocol for Eimeria nieschulzi (Apikomplexa, Coccidia).

Large numbers of sporozoites are a crucial prerequisite for in vitro experiments with Eimeria species. There are no protocols to obtain high amounts of vital purified sporozoites of Eimeria nieschulzi; therefore, an improved excystation protocol is urgently needed. Most excystation procedures for Eimeria oocysts use a mechanical disruption method for the release of sporocysts, assuming that oocyst disruption of Eimeria does not require enzymes (proteases). However, rodent Eimeria oocysts are susceptible to pepsin digestion (Kowalik S, Zahner H (1999) Eimeria separata: method for the excystation of sporozoites. Parasitol Res 85:496-499). Here, we describe a method that combines enzymatic treatment of oocyst walls before the mechanical disruption with glass beads. Using this protocol, we achieved an up to fivefold increase of free viable sporozoites of E. nieschulzi and could significantly shorten the time of excystation. These results confirm the assumption that rodent Eimeria species, in contrast to Eimeria species of birds, possess protease sensitive oocysts.

Excystation of Eimeria tenella sporozoites impaired by antibody recognizing gametocyte/oocyst antigens GAM22 and GAM56.

Eimeria tenella is the causative agent of coccidiosis in poultry. Infection of the chicken intestine begins with ingestion of sporulated oocysts releasing sporocysts, which in turn release invasive sporozoites. The monoclonal antibody E2E5 recognizes wall-forming body type II (WFBII) in gametocytes and the WFBII-derived inner wall of oocysts. Here we describe that this antibody also binds to the stieda body of sporocysts and significantly impairs in vitro excystation of sporozoites. Using affinity chromatography and protein sequence analysis, E2E5 is shown to recognize EtGAM56, the E. tenella ortholog of the Eimeria maxima gametocyte-specific GAM56 protein. In addition, this antibody was used to screen a genomic phage display library presenting E. tenella antigens as fusion proteins with the gene VIII product on the surfaces of phagemid particles and identified the novel 22-kDa histidine- and proline-rich protein EtGAM22. The Etgam22 mRNA is expressed predominantly at the gametocyte stage, as detected by Northern blotting. Southern blot analysis in combination with data from the E. tenella genome project revealed that Etgam22 is an intronless multicopy gene, with approximately 12 to 22 copies in head-to-tail arrangement. Conspicuously, Etgam56 is also intronless and is localized adjacent to another gam56-like gene, Etgam59. Our data suggest that amplification is common for genes encoding oocyst wall proteins.

Microsporidia-insect host interactions: teratoid sporogony at the sites of host tissue melanization.

Microsporidia Paranosema locustae and Paranosema grylli infect fat bodies of orthopteran hosts Locusta migratoria and Gryllus bimaculatus, respectively, and cause formation of nodules consisting of deposits of melanin around heavily infected cells. Both species sporadically produce enlarged or malformed (teratoid) spores as a result of abnormal sporogony. Proportions of teratospores within melanized nodules were 6-10 times higher than in surrounding non-melanized tissues. The increased numbers of teratoid microsporidian spores within melanized regions may indicate the deteriorating effect of melanin metabolites on spore morphogenesis.

New comprehension of the apicoplast of Sarcocystis by transmission electron tomography.

BACKGROUND INFORMATION: Apicomplexan parasites (like Plasmodium, Toxoplasma, Eimeria and Sarcocystis) contain a distinctive organelle, the apicoplast, acquired by a secondary endosymbiotic process analogous to chloroplasts and mitochondria. The apicoplast is essential for long-term survival of the parasite. This prokaryotic origin implies that molecular and metabolic processes in the apicoplast differ from those of the eukaryotic host cells and therefore offer options for specific chemotherapeutic treatment. We studied the apicoplast in high-pressure frozen and freeze-substituted cysts of Sarcocystis sp. from roe deer (Capreolus capreolus) to get better insight in apicoplast morphology. RESULTS AND CONCLUSIONS: We observed that the apicoplast contains four continuous membranes. The two inner membranes have a circular shape with a constant distance from each other and large-sized protein complexes are located between them. The two outer membranes have irregular shapes. The periplastid membrane also contains large-sized protein complexes, while the outer membrane displays protuberances into the parasite cytoplasm. In addition, it is closely associated with the endoplasmic reticulum by 'contact sites'.

Plastid segregation and cell division in the apicomplexan parasite Sarcocystis neurona.

Apicomplexan parasites harbor a secondary plastid that is essential to their survival. Several metabolic pathways confined to this organelle have emerged as promising parasite-specific drug targets. The maintenance of the organelle and its genome is an equally valuable target. We have studied the replication and segregation of this important organelle using the parasite Sarcocystis neurona as a cell biological model. This model system makes it possible to differentiate and dissect organellar growth, fission and segregation over time, because of the parasite's peculiar mode of cell division. S. neurona undergoes five cycles of chromosomal replication without nuclear division, thus yielding a cell with a 32N nucleus. This nucleus undergoes a sixth replication cycle concurrent with nuclear division and cell budding to give rise to 64 haploid daughter cells. Interestingly, intranuclear spindles persist throughout the cell cycle, thereby providing a potential mechanism to organize chromosomes and organelles in an organism that undergoes dramatic changes in ploidy. The development of the plastid mirrors that of the nucleus, a continuous organelle, which grows throughout the parasite's development and shows association with all centrosomes. Pharmacological ablation of the parasite's multiple spindles demonstrates their essential role in the organization and faithful segregation of the plastid. By using several molecular markers we have timed organelle fission to the last replication cycle and tied it to daughter cell budding. Finally, plastids were labeled by fluorescent protein expression using a newly developed S. neurona transfection system. With these transgenic parasites we have tested our model in living cells employing laser bleaching experiments.

Analysis of the Sarcocystis neurona microneme protein SnMIC10: protein characteristics and expression during intracellular development.

Sarcocystis neurona, an apicomplexan parasite, is the primary causative agent of equine protozoal myeloencephalitis. Like other members of the Apicomplexa, S. neurona zoites possess secretory organelles that contain proteins necessary for host cell invasion and intracellular survival. From a collection of S. neurona expressed sequence tags, we identified a sequence encoding a putative microneme protein based on similarity to Toxoplasma gondii MIC10 (TgMIC10). Pairwise sequence alignments of SnMIC10 to TgMIC10 and NcMIC10 from Neospora caninum revealed approximately 33% identity to both orthologues. The open reading frame of the S. neurona gene encodes a 255 amino acid protein with a predicted 39-residue signal peptide. Like TgMIC10 and NcMIC10, SnMIC10 is predicted to be hydrophilic, highly alpha-helical in structure, and devoid of identifiable adhesive domains. Antibodies raised against recombinant SnMIC10 recognised a protein band with an apparent molecular weight of 24 kDa in Western blots of S. neurona merozoites, consistent with the size predicted for SnMIC10. In vitro secretion assays demonstrated that this protein is secreted by extracellular merozoites in a temperature-dependent manner. Indirect immunofluorescence analysis of SnMIC10 showed a polar labelling pattern, which is consistent with the apical position of the micronemes, and immunoelectron microscopy provided definitive localisation of the protein to these secretory organelles. Further analysis of SnMIC10 in intracellular parasites revealed that expression of this protein is temporally regulated during endopolygeny, supporting the view that micronemes are only needed during host cell invasion. Collectively, the data indicate that SnMIC10 is a microneme protein that is part of the excreted/secreted antigen fraction of S. neurona. Identification and characterisation of additional S. neurona microneme antigens and comparisons to orthologues in other Apicomplexa could provide further insight into the functions that these proteins serve during invasion of host cells.

Monoclonal antibodies specific for the two types of wall-forming bodies of Eimeria tenella macrogametes (Coccidia, Apicomplexa).

Two monoclonal antibodies (mAbs) raised against the macrogamonts of Eimeria tenella identified antigens located in the wall-forming bodies of type I (WF I) and type II (WF II) by indirect immunofluorescence and by immunoelectron microscopy. With these mAbs, the involvement of both types of wall-forming body at the protein level in the formation of the inner and outer oocyst walls of E. tenella was shown by indirect immunofluorescence assay. On Western blots of pure macrogamont, mAb E1D8 against WF I reacted with a series of bands between 42 kDa and 105 kDa. In pure, unsporulated extract, this mAb recognized a complex of bands between 26 kDa and 153 kDa. mAb E2E5 against WF II, on Western blots of pure extract of macrogamonts, recognized an antigen of 51 kDa. Later in the development, after the formation of the inner oocyst wall, mAb E2E5 reacted with three polypeptide of 23, 25 and 30 kDa. Proteolytic processing may be forwarded as the mechanism regulating the distinct regulation protein involved in the oocyst wall.