Eimeria maxima TRAP family protein EmTFP250: subcellular localisation and induction of immune responses by immunisation with a recombinant C-terminal derivative

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.     

EtMIC4: a microneme protein from Eimeria tenella that contains tandem arrays of epidermal growth factor-like repeats and thrombospondin type-I repeats

Micronemes are specialised secretory organelles that release their proteins by a stimulus-coupled exocytosis that occurs when apicomplexan parasites make contact with target host cells. These proteins play crucial roles in motility and invasion, most likely by mediating adhesion between parasite and host cell surfaces and facilitating the transmission of dynamic forces generated by the parasite actinomyosin cytoskeleton. Members of the TRAP family of microneme proteins are characterised by having extracellular domains containing one or more types of cysteine-rich, adhesive modules, highly-conserved transmembrane regions and cytosolic tails that contain one or more tyrosines, stretches of acidic residues and a single tryptophan. In this paper, we describe a novel member of the TRAP family, EtMIC4, a 218 kDa microneme protein from Eimeria tenella. EtMIC4 contains 31 epidermal growth factor (EGF) modules, 12 thrombospondin type-1 (TSP-1) modules and a highly acidic, proline and glycine-rich region in its extracellular region, plus the conserved transmembrane and cytosolic tail. Like EtMIC1, another TRAP family member from E. tenella, EtMIC4 is expressed in sporozoites and all the merozoite stages of the parasite, suggesting that this parasite has a strong requirement for TSP-1 modules. Unlike the other microneme proteins so far studied in E. tenella, EtMIC4 appears to be found constitutively on the sporozoite surface as well as within the micronemes.     

Characterization of monoclonal antibodies that recognize the Eimeria tenella microneme protein MIC2

The apicomplexan pathogens of Eimeria cause coccidiosis, an intestinal disease of chickens, which has a major economic impact on the poultry industry. Members of the Apicomplexa share an assortment of unique secretory organelles (rhoptries, micronemes and dense granules) that mediate invasion of host cells and formation and modification of the parasitophorous vacuole. Among these, microneme protein 2 from Eimeria tenella(EtMIC2) has a putative function in parasite adhesion to the host cell to initiate the invasion process. To investigate the role of EtMIC2 in host parasite interactions, the production and characterization of 12 monoclonal antibodies (mabs) produced against recombinant EtMIC2 proteins is described. All mabs reacted with molecules belonging to the apical complex of sporozoites and merozoites of E. tenella, E. acervulina and E. maxima in an immunofluorescence assay. By Western blot analysis, the mabs identified a developmentally regulated protein of 42 kDa corresponding to EtMIC 2 and cross-reacted with proteins in developmental stages of E. acervulina. Collectively, these mabs are useful tools for the detailed investigation of the characterization of EtMIC2 related proteins in Eimeria species.     

Identification and characterization of a novel Neospora caninum immune mapped protein 1

Immune mapped protein 1 (IMP1) is a newly discovered protein in Eimeria maxima. It is recognized as a potential vaccine candidate against E. maxima and a highly conserved protein in apicomplexan parasites. Although the Neospora caninum IMP1 (NcIMP1) orthologue of E. maxima IMP1 was predicted in the N. caninum genome, it was still not identified and characterized. In this study, cDNA sequence encoding NcIMP1 was cloned by RT-PCR from RNA isolated from Nc1 tachyzoites. NcIMP1 was encoded by an open reading frame of 1182 bp, which encoded a protein of 393 amino acids with a predicted molecular weight of 42.9 kDa. Sequence analysis showed that there was neither a signal peptide nor a transmembrane region present in the NcIMP1 amino acid sequence. However, several kinds of functional protein motifs, including an N-myristoylation site and a palmitoylation site were predicted. Recombinant NcIMP1 (rNcIMP1) was expressed in Escherichia coli and then purified rNcIMP1 was used to prepare specific antisera in mice. Mouse polyclonal antibodies raised against the rNcIMP1 recognized an approximate 43 kDa native IMP1 protein. Immunofluorescence analysis showed that NcIMP1 was localized on the membrane of N. caninum tachyzoites. The N-myristoylation site and the palmitoylation site were found to contribute to the localization of NcIMP1. Furthermore, the rNcIMP1-specific antibodies could inhibit cell invasion by N. caninum tachyzoites in vitro. All the results indicate that NcIMP1 is likely to be a membrane protein of N. caninum and may be involved in parasite invasion.     

The microneme proteins EtMIC4 and EtMIC5 of Eimeria tenella form a novel, ultra-high molecular mass protein complex that binds target host cells

Eimeria tenella, in common with other parasitic protozoa of the phylum Apicomplexa, invades host cells using an actinomyosin-powered "glideosome" complex and requires the secretion of adhesive proteins from the microneme organelles onto the parasite surface. Microneme proteins of E. tenella include EtMIC4, a transmembrane protein that has multiple thrombospondin type I domains and calcium-binding epidermal growth factor-like domains in its extracellular domain, and EtMIC5, a soluble protein composed of 11 tandemly repeated domains that belong to the plasminogen-apple-nematode superfamily. We show here that EtMIC4 and EtMIC5 interact to form an oligomeric, ultrahigh molecular mass protein complex. The complex was purified from lysed parasites by non-denaturing techniques, and the stoichiometry was shown to be [EtMIC4](2):[EtMIC5](1), with an octamer of EtMIC4 bound non-covalently to a tetramer of EtMIC5. The complex is formed within the parasite secretory pathway and is maintained after secretion onto the surface of the parasite. The purified complex binds to a number of epithelial cell lines in culture. Identification and characterization of this complex contributes to an overall understanding of the role of multimolecular protein complexes in specific interactions between pathogens and their hosts during infection.     

Domains of invasion organelle proteins from apicomplexan parasites are homologous with the Apple domains of blood coagulation factor XI and plasma pre-kallikrein and are members of the PAN module superfamily

Micronemes are specialised organelles, found in all apicomplexan parasites, which secrete molecules that are essential for parasite attachment to and invasion of host cells. Regions of several microneme proteins have sequence similarity to the Apple domains (A-domains) of blood coagulation factor XI (FXI) and plasma pre-kallikrein (PK). We have used mass spectrometry on a recombinant-expressed, putative A-domain from the microneme protein EtMIC5 from Eimeria tenella, to demonstrate that three intramolecular disulphide bridges are formed. These bridges are analogous to those that stabilise A-domains in FXI and PK. The data confirm that the apicomplexan domains are structural homologues of A-domains and are therefore novel members of the PAN module superfamily, which also includes the N-terminal domains of members of the plasminogen/hepatocyte growth factor family. The role of A-domains/PAN modules in apicomplexan parasites is not known, but their presence in the microneme suggests that they may be important for mediating protein-protein or protein-carbohydrate interactions during parasite attachment and host cell invasion.     

Two separate, conserved acidic amino acid domains within the Toxoplasma gondii MIC2 cytoplasmic tail are required for parasite survival

Apicomplexan parasites rely on actin-based motility to drive host cell invasion. Motility and invasion also require thrombospondin-related anonymous protein (TRAP) adhesins, which are secreted apically and translocated to the posterior end of the parasite before they are shed by the activity of a rhomboid protease. TRAP orthologs, including Toxoplasma gondii MIC2 (microneme protein 2), possess a short cytoplasmic tail, which is essential for motility. Previous studies have shown that aldolase forms a critical bridge between actin filaments and the cytoplasmic domains of MIC2 and TRAP. The cytoplasmic tails of TRAP family members harbor a conserved penultimate tryptophan, which is essential for aldolase binding, and clustered acidic residues. Herein, we determined the role of the conserved acidic residues by using alanine point mutants to investigate aldolase binding in vitro and to test functionality in the parasite. Our studies revealed two separate acidic residue clusters in the cytoplasmic domain of MIC2 that are essential for parasite survival. One region, located at the extreme C terminus, is required for the direct interaction with aldolase, whereas the second upstream acidic region is not necessary for aldolase binding but is nonetheless essential to parasite survival. Both acidic domains are conserved throughout TRAP orthologs, implicating a central role for these motifs in apicomplexan motility.     

The development of the macrogamete and oocyst wall in Eimeria maxima: immuno-light and electron microscopy

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.     

Identifying and characterising the Plasmodium falciparum merozoite surface protein 10 Plasmodium vivax homologue

Plasmodium vivax malaria is one of the most prevalent parasitic diseases in Asia and Latin-America. The difficulty of maintaining this parasite culture in vitro has hampered identifying and characterising proteins implied in merozoite invasion of red blood cells. We have been able to identify an open reading frame in P. vivax encoding the Plasmodium falciparum merozoite surface protein 10 homologous protein using the partial sequences from this parasite's genome reported during 2004. This new protein contains 479 amino-acids, two epidermal growth factor-like domains, hydrophobic regions at the N- and C-termini, being compatible with a signal peptide and a glycosylphosphatidylinositol anchor site, respectively. The protein is expressed during the parasite's asexual stage and is recognised by polyclonal sera in parasite lysate using Western blot. P. vivax-infected patients' sera highly recognised recombinant protein by ELISA.     

The layered fold of the TSR domain of P. falciparum TRAP contains a heparin binding site

Thrombospondin-related anonymous protein, TRAP, has a critical role in the hepatocyte invasion step of Plasmodium sporozoites, the transmissible form of the parasite causing malaria. The extracellular domains of this sporozoite surface protein interact with hepatocyte surface receptors whereas its intracellular domain acts as a link to the sporozoite actomyosin motor system. Liver heparan sulfate proteoglycans have been identified as potential ligands for TRAP. Proteoglycan binding has been associated with the A- and TSR domains of TRAP. We present the solution NMR structure of the TSR domain of TRAP and a chemical shift mapping study of its heparin binding epitope. The domain has an elongated structure stabilized by an array of tryptophan and arginine residues as well as disulfide bonds. The fold is very similar to those of thrombospondin type-1 (TSP-1) and F-spondin TSRs. The heparin binding site of TRAP-TSR is located in the N-terminal half of the structure, the layered side chains forming an integral part of the site. The smallest heparin fragment capable of binding to TRAP-TSR is a tetrasaccharide.     

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.     

A novel serralysin metalloprotease from Deinococcus radiodurans

A hypothetical protein (DR2310) from the radiation resistant organism Deinococcus radiodurans harbors highly conserved Zn(+2)-binding (HEXXH) domain and Met-turn (SVMSY), characteristic of the serralysin family of secreted metalloproteases from Gram negative bacteria. Deletion mutagenesis of DR2310 confirmed that the ORF is expressed in Deinococcus radiodurans as a secreted protease of 85 kDa. Biochemical analysis revealed DR2310 to be a Ca(+2) and Zn(+2)-requiring metalloprotease. Unique features such as a long N-terminus, replacement of the highly conserved C-terminal glycine rich Ca(+2)-binding repeats with a single N-terminal aspartate rich eukaryotic thrombospondin type-3 Ca(+2)-binding repeat and absence of C-terminal secretion signals make it a novel member of serralysin family. This is the first report of a functional serralysin family metalloprotease from a Gram positive organism.     

MORN1 has a conserved role in asexual and sexual development across the apicomplexa

The gene encoding the membrane occupation and recognition nexus protein MORN1 is conserved across the Apicomplexa. In Toxoplasma gondii, MORN1 is associated with the spindle poles, the anterior and posterior rings of the inner membrane complex (IMC). The present study examines the localization of MORN1 during the coccidian development of T. gondii and three Eimeria species (in the definitive host) and erythrocytic schizogony of Plasmodium falciparum. During asexual proliferation, MORN1 is associated with the posterior ring of the IMCs of the multiple daughters forming during T. gondii endopolygeny and schizogony in Eimeria and P. falciparum. Furthermore, the expression of P. falciparum MORN1 protein peaked in late schizogony. These data fit a model with a conserved role for MORN1 during IMC assembly in all variations of asexual development. An important new observation is the reactivity of MORN1 antibody with certain sexual stages in T. gondii and Eimeria species. Here MORN1 is organized as a ring-like structure where the microgametes bud from the microgametocyte while in mature microgametes it is present near the flagellar basal bodies and mitochondrion. These observations suggest a conserved role for MORN1 in both asexual and sexual development across the Apicomplexa.     

From hepatopancreas to ovary: molecular characterization of a shrimp vitellogenin receptor involved in the processing of vitellogenin

We report the first cloning and characterization of cDNA encoding a putative vitellogenin (Vg) receptor (VgR) from the shrimp, Penaeus monodon. The shrimp VgR cDNA is 6.8 kb; the deduced protein has 1943 amino acids with a molecular weight of 211 kDa. VgR is ovary specific and consists of conserved cysteine-rich domains, epidermal growth factor-like domains, and YWTD motifs similar to the low-density lipoprotein, very low-density lipoprotein, and VgR of insects and vertebrates. VgR expression level in the ovary is low during early vitellogenesis and increases to maximum levels in females with a gonadosomatic index of 3-4, presumably when needed for receptor-mediated endocytosis during the rapid phase of extraovarian Vg production by the hepatopancreas. A peptide from the C-terminal end of VgR was synthesized for antibody production. Anti-VgR antibody recognized an ovarian membrane protein, and the level of this protein was high when extraovarian production of Vg reached peak levels. By immunohistochemical analysis, VgR was detected strongly in the membranes of larger oocytes. VgR expression was knocked down after the shrimp were injected with VgR double-stranded RNA, leading to a decrease in VgR protein content in the ovary, but an increase in the hemolymph level of Vg. This study represents the first report of the functional analysis of a putative VgR in a crustacean.     

The Thrombospondin-related Protein Family of Apicomplexan Parasites: The Gears of the Cell Invasion Machinery

A number of severe diseases of medical and veterinary importance are caused by parasites of the phylum Apicomplexa. These parasites invade host cells using similar subcellular structures, organelles and molecular species. Proteins containing one or more copies of the type I repeat of human platelet thrombospondin (TSP1), are crucial components of both locomotion and invasion machinery. Members of this family have been identified in Eimeria tenella, E. maxima, Toxoplasma gondii, Cryptosporidium parvum and in all Plasmodium species so far analysed. Here, Andrea Crisanti and colleagues discuss the structure, localization and current understanding of the function of TSP family members in the invasion of target cells by apicomplexan parasites.     

Purification, characterization and cloning of antiviral/ribosome inactivating protein from Amaranthus tricolor leaves

An antiviral protein (AVP), imparting high level of resistance against sunnhemp rosette virus (SRV) was purified from the dried leaves of Amaranthus tricolor. The purified protein (AAP-27) exhibited approximately 98% inhibition of local lesion formation at a concentration range of approximately 30 microg ml(-1). The protein was found to be highly basic glycoprotein monomer (pI approximately 9.8) of Mr 27 kDa, with neutral sugar content of 4%. The purified protein exhibited N-glycosidase and RNase activities. We have also isolated full-length cDNA clone, encoding this protein designated as A. tricolor antiviral protein-1 (AAP-1). Two primers, one designed on the basis of N-terminal sequence of the purified protein and the other from the conserved active peptides of other AVPs/RIPs were used for PCR amplification of double stranded cDNA, isolated from the leaves of A. tricolor. The amplified fragment was used as a probe for library screening. The isolated full-length cDNA consisted of 1058 nucleotides with an open reading frame encoding a polypeptide of 297 amino acids. The deduced amino acid sequence of AAP-1 has a putative active domain conserved in other AVPs/RIPs and shows varying homology to the RIPs from other plant species.     

Defining the protein repertoire of microneme secretory organelles in the apicomplexan parasite Eimeria tenella

The apicomplexan pathogen Eimeria causes coccidiosis, an intestinal disease of chickens, which has a major welfare and economic impact on the poultry industry. There is an urgent need to identify molecules that are rational targets for drug design and novel vaccines against coccidiosis. Apicomplexan secretory organelles, including micronemes and rhoptries, are essential for invasion of the host intestinal epithelium and establishment of parasitism. However, relatively little is known about the precise molecular function of these organelles, partly because few organelle proteins have been characterized. In this study, proteomics tools have been harnessed to define the protein repertoire of micronemes. Purified microneme proteins from Eimeria tenella sporozoites were excised from two-dimensional (2-D) gels and analyzed using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and chemically assisted fragmentation (CAF)-MALDI with de novo sequencing. Peptide mass profiles were searched against the NCBI non-redundant (nr) database and against Eimeria-specific databases using the Mascot search algorithm, resulting in the identification of 37 of 96 spots excised from the 2-D gels. In addition, we have found CAF-MALDI to be a useful adjunct for identifying proteins, without the need for tandem MS. This global approach to protein characterization will be vital to gain greater understanding of the processes involved in apicomplexan host cell invasion.     

Eimeria acervulina: cloning of a cDNA encoding an immunogenic region of several related merozoite surface and rhoptry proteins.

A cDNA encoding a recombinant Eimeria acervulina antigen, designated EAMZp30-47, that contains an epitope shared among several surface and rhoptry proteins of merozoites was characterized. The respective parasite proteins are between 30 and 47 kDa as revealed by immunostaining of nitrocellulose membrane containing extracts of 125I-labeled merozoites. As indicated by immunofluorescence and immunoelectron microscopic staining, the reactive epitope was localized to both the surface membrane and the internal rhoptries of this asexual stage of the parasite. The recombinant beta-galactosidase fusion protein EAMZp30-47 is 130 kDa, thus representing 15 kDa or 30-50% of the respective parasite protein. Purified EAMZp30-47 stimulates T cells from E. acervulina-immune inbred chickens, but is not recognized by immune chicken serum, suggesting that T cell and not B cell epitopes recognized by the host immune system during a natural infection are present on the recombinant protein. Northern and Southern blot hybridization experiments indicated that expression of EAMZp30-47 is restricted to the merozoite stage of the parasite and the gene occurs as a single copy sequence within the genome.