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Since Nicolle, Manceaux and Splendore first described Toxoplasma gondii as a parasite of rodents and rabbits in the early 20th century, a diverse and vigorous research community has been built around studying this fascinating intracellular parasite. In addition to its importance as a pathogen of humans, livestock and wildlife, modern researchers are attracted to T. gondii as a facile experimental system to study many aspects of evolutionary biology, cellular biology, host-microbe interactions, and host immunity. For new researchers entering the field, the extensive literature describing the biology of the parasite, and the interactions with its host, can be daunting. In this review, we examine four foundational studies that describe various aspects of T. gondii biology, presenting a 'journal club'-style analysis of each. We have chosen a paper that established the beguiling life cycle of the parasite (Hutchison et al., 1971), a paper that described key features of its cellular biology that the parasite shares with related organisms (Gustafson et al., 1954), a paper that characterised the origin of the unique compartment in which the parasite resides within host cells (Jones and Hirsch, 1972), and a paper that established a key mechanism in the host immune response to parasite infection (Pfefferkorn, 1984). These interesting and far-reaching studies set the stage for subsequent research into numerous facets of parasite biology. As well as providing new researchers with an entry point into the literature surrounding the parasite, revisiting these studies can remind us of the roots of our discipline, how far we have come, and the new directions in which we might head.

Since Nicolle, Manceaux and Splendore first described Toxoplasma gondii as a parasite of rodents and rabbits in the early 20th century, a diverse and vigorous research community has been built around studying this fascinating intracellular parasite. In addition to its importance as a pathogen of humans, livestock and wildlife, modern researchers are attracted to T. gondii as a facile experimental system to study many aspects of evolutionary biology, cellular biology, host-microbe interactions, and host immunity. For new researchers entering the field, the extensive literature describing the biology of the parasite, and the interactions with its host, can be daunting. In this review, we examine four foundational studies that describe various aspects of T. gondii biology, presenting a 'journal club'-style analysis of each. We have chosen a paper that established the beguiling life cycle of the parasite (Hutchison et al., 1971), a paper that described key features of its cellular biology that the parasite shares with related organisms (Gustafson et al., 1954), a paper that characterised the origin of the unique compartment in which the parasite resides within host cells (Jones and Hirsch, 1972), and a paper that established a key mechanism in the host immune response to parasite infection (Pfefferkorn, 1984). These interesting and far-reaching studies set the stage for subsequent research into numerous facets of parasite biology. As well as providing new researchers with an entry point into the literature surrounding the parasite, revisiting these studies can remind us of the roots of our discipline, how far we have come, and the new directions in which we might head.

Stable isotopes are ideal labels for studying biological processes because they have little or no effect on the biochemical properties of target molecules. The NanoSIMS is a tool that can image the distribution of stable isotope labels with up to 50 nm spatial resolution and with good quantitation. This combination of features has enabled several groups to undertake significant experiments on biological problems in the last decade. Combining the NanoSIMS with other imaging techniques also enables us to obtain not only chemical information but also the structural information needed to understand biological processes. This article describes the methodologies that we have developed to correlate atomic force microscopy and backscattered electron imaging with NanoSIMS experiments to illustrate the imaging of stable isotopes at molecular, cellular, and tissue scales. Our studies make it possible to address 3 biological problems: (1) the interaction of antimicrobial peptides with membranes; (2) glutamine metabolism in cancer cells; and (3) lipoprotein interactions in different tissues.

The basic organisation of the endomembrane system is conserved in all eukaryotes and comparative genome analyses provides compelling evidence that the endomembrane system of the last common eukaryotic ancestor (LCEA) is complex with many genes required for regulated traffic being present. Although apicomplexan parasites, causative agents of severe human and animal diseases, appear to have only a basic set of trafficking factors such as Rab-GTPases, they evolved unique secretory organelles (micronemes, rhoptries and dense granules) that are sequentially secreted during invasion of the host cell. In order to define the secretory pathway of apicomplexans, we performed an overexpression screen of Rabs in Toxoplasma gondii and identified Rab5A and Rab5C as important regulators of traffic to micronemes and rhoptries. Intriguingly, we found that not all microneme proteins traffic depends on functional Rab5A and Rab5C, indicating the existence of redundant microneme targeting pathways. Using two-colour super-resolution stimulated emission depletion (STED) we verified distinct localisations of independent microneme proteins and demonstrate that micronemal organelles are organised in distinct subsets or subcompartments. Our results suggest that apicomplexan parasites modify classical regulators of the endocytic system to carryout essential parasite-specific roles in the biogenesis of their unique secretory organelles.

The oocyst wall of coccidian parasites is a robust structure that is resistant to a variety of environmental and chemical insults. This resilience allows oocysts to survive for long periods, facilitating transmission from host to host. The wall is bilayered and is formed by the sequential release of the contents of two specialized organelles - wall forming body 1 and wall forming body 2 - found in the macrogametocyte stage of Coccidia. The oocyst wall is over 90% protein but few of these proteins have been studied. One group is cysteine-rich and may be presumed to crosslink via disulphide bridges, though this is yet to be investigated. Another group of wall proteins is rich in tyrosine. These proteins, which range in size from 8-31 kDa, are derived from larger precursors of 56 and 82 kDa found in the wall forming bodies. Proteases may catalyze processing of the precursors into tyrosine-rich peptides, which are then oxidatively crosslinked in a reaction catalyzed by peroxidases. In support of this hypothesis, the oocyst wall has high levels of dityrosine bonds. These dityrosine crosslinked proteins may provide a structural matrix for assembly of the oocyst wall and contribute to its resilience.

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. © 2009 Australian Society for Parasitology Inc.

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. © 2009 Australian Society for Parasitology Inc.

The oocyst wall of coccidian parasites is a robust structure that is resistant to a variety of environmental and chemical insults. This resilience allows oocysts to survive for long periods, facilitating transmission from host to host. The wall is bilayered and is formed by the sequential release of the contents of two specialized organelles - wall forming body 1 and wall forming body 2 - found in the macrogametocyte stage of Coccidia. The oocyst wall is over 90% protein but few of these proteins have been studied. One group is cysteine-rich and may be presumed to crosslink via disulphide bridges, though this is yet to be investigated. Another group of wall proteins is rich in tyrosine. These proteins, which range in size from 8-31 kDa, are derived from larger precursors of 56 and 82 kDa found in the wall forming bodies. Proteases may catalyze processing of the precursors into tyrosine-rich peptides, which are then oxidatively crosslinked in a reaction catalyzed by peroxidases. In support of this hypothesis, the oocyst wall has high levels of dityrosine bonds. These dityrosine crosslinked proteins may provide a structural matrix for assembly of the oocyst wall and contribute to its resilience.

Coccidian parasites are transmitted between hosts by the ingestion of food or water contaminated with oocysts, followed by the release of infectious sporozoites and invasion of the gastro-intestinal tract. In the external environment, sporozoites are protected from desiccation and chemical disinfection by the oocyst wall. This unique structure guarantees successful disease transmission and is as vital to the coccidian parasite as the exoskeleton is to insects - without it they would die. Here, we revisit the early work and combine it with newer molecular data to describe our present understanding of the coccidian oocyst wall. © 2006 Elsevier Ltd. All rights reserved.

Coccidian parasites are transmitted between hosts by the ingestion of food or water contaminated with oocysts, followed by the release of infectious sporozoites and invasion of the gastro-intestinal tract. In the external environment, sporozoites are protected from desiccation and chemical disinfection by the oocyst wall. This unique structure guarantees successful disease transmission and is as vital to the coccidian parasite as the exoskeleton is to insects - without it they would die. Here, we revisit the early work and combine it with newer molecular data to describe our present understanding of the coccidian oocyst wall. © 2006 Elsevier Ltd. All rights reserved.

EmTFP250 is a high molecular mass, asexual stage antigen from Eimeria maxima strongly associated with maternally derived immunity to this protozoan parasite in hatchling chickens. Cloning and sequence analysis has predicted the antigen to be a novel member of the thrombospondin-related anonymous protein (TRAP) family of apicomplexan parasites. Members of the TRAP family are microneme proteins and are associated with host cell invasion and apicomplexan gliding motility. In order to assess the immunogenicity of EmTFP250, a C-terminal derivative encoding a low complex, hydrophilic region and putative transmembrane domain/cytosolic tail was expressed in a bacterial host system. The recombinant protein was used to immunise mice and chickens and found to induce strong IgG responses in both animal models as determined by specific ELISAs. Using Western blotting, protective maternal IgG antibodies previously shown to recognise native EmTFP250 recognised the recombinant protein and, in addition, antibodies raised against the recombinant protein were shown to recognise native EmTFP250. Localisation studies employing immuno-light microscopy and immuno-electron microscopy showed that antibodies to the recombinant protein specifically labeled micronemes within merozoites of E. maxima. Furthermore, antibodies to the recombinant EmTFP250 derivative showed similar labeling of micronemes within merozoites of Eimeria tenella. This study is further suggestive of a functional importance for EmTFP250 and underscores its potential as a candidate for a recombinant vaccine targeting coccidiosis in chickens. © 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

EmTFP250 is a high molecular mass, asexual stage antigen from Eimeria maxima strongly associated with maternally derived immunity to this protozoan parasite in hatchling chickens. Cloning and sequence analysis has predicted the antigen to be a novel member of the thrombospondin-related anonymous protein (TRAP) family of apicomplexan parasites. Members of the TRAP family are microneme proteins and are associated with host cell invasion and apicomplexan gliding motility. In order to assess the immunogenicity of EmTFP250, a C-terminal derivative encoding a low complex, hydrophilic region and putative transmembrane domain/cytosolic tail was expressed in a bacterial host system. The recombinant protein was used to immunise mice and chickens and found to induce strong IgG responses in both animal models as determined by specific ELISAs. Using Western blotting, protective maternal IgG antibodies previously shown to recognise native EmTFP250 recognised the recombinant protein and, in addition, antibodies raised against the recombinant protein were shown to recognise native EmTFP250. Localisation studies employing immuno-light microscopy and immuno-electron microscopy showed that antibodies to the recombinant protein specifically labeled micronemes within merozoites of E. maxima. Furthermore, antibodies to the recombinant EmTFP250 derivative showed similar labeling of micronemes within merozoites of Eimeria tenella. This study is further suggestive of a functional importance for EmTFP250 and underscores its potential as a candidate for a recombinant vaccine targeting coccidiosis in chickens. © 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

We have identified, and followed the development of three macrogamete organelles involved in the formation of the oocyst wall of Eimeria maxima. The first were small lucent vacuoles that cross-reacted with antibodies to the apple domains of the Toxoplasma gondii microneme protein 4. They appeared early in development and were secreted during macrogamete maturation to form an outer veil and were termed veil forming bodies. The second were the wall forming bodies type 1, large, electron dense vacuoles that stained positively only with antibodies raised to an enriched preparation of the native forms of 56 (gam56), 82 (gam82) and 230 kDa (gam230) gametocyte antigens (termed anti-APGA). The third were the wall forming bodies type 2, which appeared before the wall forming bodies type 1 but remain enclosed within the rough endoplasmic reticulum and stained positively with antibodies raised to recombinant versions of gam56 (anti-gam56), gam82 (anti-gam82) and gam230 (anti-gam230) plus anti-APGA. At the initiation of oocyst wall formation, the anti-T. gondii microneme protein 4 positive outer veil detached from the surface. The outer layer of the oocyst wall was formed by the release of the contents of wall forming bodies type 1 at the surface to form an electron dense, anti-APGA positive layer. The wall forming bodies type 2 appeared, subsequently, to give rise to the electron lucent inner layer. Thus, oocyst wall formation in E. maxima represents a sequential release of the contents of the veil forming bodies, wall forming bodies types 1 and 2 and this may be controlled at the level of the rough endoplasmic reticulum/Golgi body. © 2003 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

We have identified, and followed the development of three macrogamete organelles involved in the formation of the oocyst wall of Eimeria maxima. The first were small lucent vacuoles that cross-reacted with antibodies to the apple domains of the Toxoplasma gondii microneme protein 4. They appeared early in development and were secreted during macrogamete maturation to form an outer veil and were termed veil forming bodies. The second were the wall forming bodies type 1, large, electron dense vacuoles that stained positively only with antibodies raised to an enriched preparation of the native forms of 56 (gam56), 82 (gam82) and 230 kDa (gam230) gametocyte antigens (termed anti-APGA). The third were the wall forming bodies type 2, which appeared before the wall forming bodies type 1 but remain enclosed within the rough endoplasmic reticulum and stained positively with antibodies raised to recombinant versions of gam56 (anti-gam56), gam82 (anti-gam82) and gam230 (anti-gam230) plus anti-APGA. At the initiation of oocyst wall formation, the anti-T. gondii microneme protein 4 positive outer veil detached from the surface. The outer layer of the oocyst wall was formed by the release of the contents of wall forming bodies type 1 at the surface to form an electron dense, anti-APGA positive layer. The wall forming bodies type 2 appeared, subsequently, to give rise to the electron lucent inner layer. Thus, oocyst wall formation in E. maxima represents a sequential release of the contents of the veil forming bodies, wall forming bodies types 1 and 2 and this may be controlled at the level of the rough endoplasmic reticulum/Golgi body. © 2003 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.