Multimerization of the Toxoplasma gondii MIC2 integrin-like A-domain is required for binding to heparin and human cells

Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis in humans and animals. To invade host cells, T. gondii deploys the contents of its apically oriented secretory organelles that include the micronemes. Contained within the micronemes are proteins that possess adhesive motifs resembling those found in mammalian proteins. The micronemal protein MIC2 is a member of the thrombospondin-related anonymous protein (TRAP) family of adhesive proteins, which characteristically feature at least one integrin-like A-domain. Because of its strict conservation within the family, we sought to define the role of this domain by testing the adhesive properties of recombinant MIC2 A-domain fusion proteins. Since MIC2 is found as a multimeric species in parasite lysate, we also wanted to test whether recombinant MIC2 A-domain bound to its substrate in a multimeric state. In vitro assays of binding to several different potential receptors revealed that the MIC2 A-domain binds specifically to heparin, a ubiquitous sulfated proteoglycan found in the extracellular matrix (ECM). Additional studies demonstrated that this binding is not dependent on the MIDAS site, a well-conserved divalent cation-binding motif that the MIC2 A-domain shares with its mammalian counterparts. The recombinant MIC2 A-domain bound to heparin as a high molecular weight species, as did MIC2 from parasite lysate, indicating that the recombinant A-domain mimics the binding of native MIC2. Multimerization of MIC2 may increase the number of interactions with host cell receptors, thereby forming a multivalent adhesive junction during parasite entry.     

Preparation and initial application of monoclonal antibodies that recognize Eimeria tenella microneme proteins 1 and 2

Microneme proteins (MICs) of Eimeria species are critical for motility of the parasite, identification and binding of host cell-surface proteins, invasion of host cells, and intracellular survival. The microneme protein 1 (EtMIC1) and 2 (EtMIC2) from Eimeria tenella have a putative function in parasite adhesion to the host cell to initiate an invasion process. Previous studies indicated that the EtMIC1 and EtMIC2 proteins form a complex that play roles during attachment to and penetration of the host cell. Numerous studies demonstrated that both the EtMIC1 and EtMIC2 are important microneme proteins which are abundantly expressed in sporozoites and schizogony stages. But the expression of EtMIC1 and EtMIC2 in the gametogony stage is unknown. To investigate the precise roles of EtMIC1 and EtMIC2 in host-parasite interactions and expressions in the gametogony stage of E. tenella, we generated five mouse monoclonal antibodies (MAbs) which recognize the EtMIC1 and EtMIC2 proteins and investigated expressions of EtMIC1 and EtMIC2 proteins in later endogenous developmental stages, particularly focused on the gametogony phase using the specific anti-EtMIC1 and anti-EtMIC2 MAbs produced in this work. Our results showed that both EtMIC1 and EtMIC2 proteins are expressed in all developmental stages including the gametogony stage. To our knowledge, this is the first report that the EtMIC1 and EtMIC2 proteins are expressed in the gametogony stage of E. tenella.     

Eimeria tenella microneme protein EtMIC3: identification, localisation and role in host cell infection

The gene coding for Eimeria tenella protein EtMIC3 was cloned by screening a sporozoite cDNA library with two independent monoclonal antibodies raised against the oocyst stage. The deduced sequence of EtMIC3 is 988 amino acids long. The protein presents seven repeats in tandem, with four highly conserved internal repeats and three more divergent external repeats. Each repeat is characterised by a tyrosine kinase phosphorylation site, WRCY, and a reminiscent motif of the thrombospondin1 (TSP1)-type I domain, CXXXCG. The protein EtMIC3 is localised at the apex of free parasite stages. It is not detected in the early intracellular parasite stage but is synthesised in mature schizonts. Secretion of the protein is induced when sporozoites are incubated in complete medium at 41 degrees C. Strangely enough, the two independent mAb that allow cloning of EtMIC3 interfere with parasitic growth in different ways. One is able to inhibit parasite invasion whereas the other inhibits development. Expression and localisation of the protein EtMIC3 are consistent with a protein involved in the invasion process as is expected for a microneme protein.     

Induction of secretion and surface capping of microneme proteins in Eimeria tenella

Micronemes are secretory organelles of the invasive stages of apicomplexan parasites and contain proteins that are important for parasite motility and host cell invasion. We have examined the induction of microneme secretion in the coccidian Eimeria tenella. When sporozoites were added to MDBK cells in culture, microneme proteins were secreted, capped backwards over the parasite surface and deposited onto underlying host cells from the posterior end of gliding parasites. Induction of secretion was also achieved by the addition of foetal calf serum, or purified albumin, to extracellular sporozoites. Microneme secretion per se was not dependent on parasites being able to move or to invade host cells. However, in the presence of cytochalasin D, which disrupts actin polymerisation and prevents parasite movement, microneme proteins were secreted from the apical tip but were not capped backwards over the sporozoite surface. These observations support the hypothesis that microneme proteins function as ligands which, when secreted out onto the parasite surface, form a link, either directly or indirectly, between the sub-pellicular actin–myosin cytoskeletal motor of the parasite and the surface of target host cells.     

Calcium binding activity of the epidermal growth factor-like domains of the apicomplexan microneme protein EtMIC4

Microneme proteins are secreted from apicomplexan parasites during invasion of host cells and they play crucial roles in parasite-host cell adhesion. EtMIC4 is a 240 kDa transmembrane protein from Eimeria tenella that contains 31 tandemly arranged epidermal growth factor (EGF), like repeats within its extracellular domain. The majority of these repeats have calcium binding (cb) consensus sequences. Little is known about cbEGFs in apicomplexan parasites but their presence in microneme proteins suggests that they may contribute to parasite-host interactions. To investigate the potential role of cbEGFs we have expressed and correctly refolded a cbEGF triplet from EtMIC4 (cbEGF7-9) and demonstrated that this triplet binds calcium. Circular dichroism spectroscopic analysis of cbEGF7-9 demonstrates that the molecule undergoes a gradual change in conformation with increasing levels of calcium. In the presence of calcium, the triplet becomes resistant to proteolytic degradation by a variety of proteases, a characteristic feature of cbEGF repeats from higher eukaryotic proteins, such as fibrillin, suggesting that calcium binding induces the formation of a rigid conformation. Moreover, mass spectrometric mapping of the cleavage sites that are protected by calcium shows that these sites are located both close to and distant from the calcium binding sites, indicating that protection is not due to steric hindrance by calcium ions, but rather due to the overall conformation adopted by the triplet in the presence of calcium. Thus, the tandemly-arranged cbEGF repeats within EtMIC4 provide a mechanism whereby, in the calcium-rich extracellular environment, the molecule could adopt a protease-resistant, rigid structure that could favour its interaction with host cell ligands.     

Sequence of the gene encoding an immunodominant microneme protein of Eimeria tenella.

A heterodisperse family of antigens, previously detected on sporozoites and merozoites of Eimeria tenella, has been localised to the microneme organelles within the sporozoite. Sequencing of genomic and cDNA clones shows that the gene for this antigen family contains 4 exons separated by 3 short (519, 226 and 156 nucleotides) intervening sequences and that the predicted polypeptide from the longest open reading frame has 4 structural domains. One of these contains 5 copies of the thrombospondin-like motif, previously identified in the partial sequence of the gene, which is conserved in a variety of molecules which have been demonstrated to have adhesive properties. A second domain of the polypeptide has strong similarity to a conserved region that occurs in another group of molecules which have adhesive properties, including the alpha subunits of several integrins, complement factor Bb and a number of extracellular matrix glycoproteins. Overall the antigen resembles the thrombospondin-related anonymous protein identified in the erythrocytic stage of Plasmodium falciparum. The structure of the gene supports a role for this microneme antigen in cell-cell or cell-matrix interactions.     

Family members stick together: multi-protein complexes of malaria parasites

Malaria parasites express a broad repertoire of proteins whose expression is tightly regulated depending on the life-cycle stage of the parasite and the environment of target organs in the respective host. Transmission of malaria parasites from the human to the anopheline mosquito is mediated by intraerythrocytic sexual stages, termed gametocytes, which circulate in the peripheral blood and are essential for the spread of the tropical disease. In Plasmodium falciparum, gametocytes express numerous extracellular proteins with adhesive motifs, which might mediate important interactions during transmission. Among these is a family of six secreted proteins with adhesive modules, termed PfCCp proteins, which are highly conserved throughout the apicomplexan clade. In P. falciparum, the proteins are expressed in the parasitophorous vacuole of gametocytes and are subsequently exposed on the surface of macrogametes during parasite reproduction in the mosquito midgut. One characteristic of the family is a co-dependent expression, such that loss of all six proteins occurs if expression of one member is disrupted via gene knockout. The six PfCCp proteins interact by adhesion domain-mediated binding and thus form complexes on the sexual stage surface having adhesive properties. To date, the PfCCp proteins represent the only protein family of the malaria parasite sexual stages that assembles to multimeric complexes, and only a small number of such protein complexes have so far been identified in other life-cycle stages of the parasite.     

艾美耳球虫子孢子从宿主细胞中逸出机制的初步研究

艾美耳球虫是一类重要的肠道病原,其裂殖生殖阶段的虫体逸出过程是造成畜禽肠道破坏的主要原因之一,但此逸出过程的机制仍鲜有报道。本研究以乙醇作为诱导剂研究柔嫩艾美耳球虫M2e株子孢子从宿主细胞中逸出的机制。结果显示,乙醇可诱导子孢子从MDBK细胞中逸出,此逸出过程依赖于虫体的运动能力;同时,乙醇可激发子孢子逸出相关的微线体蛋白2（Mic2）的分泌释放。进一步实验证实,螯合虫体内部钙离子明显阻断了子孢子逸出及Mic2蛋白的释放。本研究初步证实了与柔嫩艾美耳球虫逸出相关的蛋白和离子,为深入解析球虫致病的分子机制、研发新型抗球虫药物提供了新的研究方向。     

Induction of secretion and surface capping of microneme proteins in Eimeria tenella

Micronemes are secretory organelles of the invasive stages of apicomplexan parasites and contain proteins that are important for parasite motility and host cell invasion. We have examined the induction of microneme secretion in the coccidian Eimeria tenella. When sporozoites were added to MDBK cells in culture, microneme proteins were secreted, capped backwards over the parasite surface and deposited onto underlying host cells from the posterior end of gliding parasites. Induction of secretion was also achieved by the addition of foetal calf serum, or purified albumin, to extracellular sporozoites. Microneme secretion per se was not dependent on parasites being able to move or to invade host cells. However, in the presence of cytochalasin D, which disrupts actin polymerisation and prevents parasite movement, microneme proteins were secreted from the apical tip but were not capped backwards over the sporozoite surface. These observations support the hypothesis that microneme proteins function as ligands which, when secreted out onto the parasite surface, form a link, either directly or indirectly, between the sub-pellicular actin–myosin cytoskeletal motor of the parasite and the surface of target host cells.     

Fetuin-A, a hepatocyte-specific protein that binds Plasmodium berghei thrombospondin-related adhesive protein: a potential role in infectivity

Malaria infection is initiated when the insect vector injects Plasmodium sporozoites into a susceptible vertebrate host. Sporozoites rapidly leave the circulatory system to invade hepatocytes, where further development generates the parasite form that invades and multiplies within erythrocytes. Previous experiments have shown that the thrombospondin-related adhesive protein (TRAP) plays an important role in sporozoite infectivity for hepatocytes. TRAP, a typical type-1 transmembrane protein, has a long extracellular region, which contains two adhesive domains, an A-domain and a thrombospondin repeat. We have generated recombinant proteins of the TRAP adhesive domains. These TRAP fragments show direct interaction with hepatocytes and inhibit sporozoite invasion in vitro. When the recombinant TRAP A-domain was used for immunoprecipitation against hepatocyte membrane fractions, it bound to alpha2-Heremans-Schmid glycoprotein/fetuin-A, a hepatocyte-specific protein associated with the extracellular matrix. When the soluble sporozoite protein fraction was immunoprecipitated on a fetuin-A-adsorbed protein A column, TRAP bound this ligand. Importantly, anti-fetuin-A antibodies inhibited invasion of hepatocytes by sporozoites. Further, onset of malaria infection was delayed in fetuin-A-deficient mice compared to that in wild-type C57BL/6 mice when they were challenged with Plasmodium berghei sporozoites. These data demonstrate that the extracellular region of TRAP interacts with fetuin-A on hepatocyte membranes and that this interaction enhances the parasite's ability to invade hepatocytes.     

Identification of a Neospora caninum microneme protein (NcMIC1) which interacts with sulfated host cell surface glycosaminoglycans

The invasive stages of apicomplexan parasites enter their host cells through mechanisms which are largely conserved throughout the phylum. Host cell invasion is divided into two distinct events, namely, adhesion onto the host cell surface and the actual host cell entry process. The former is mediated largely through microneme proteins which are secreted at the onset of establishing contact with the host cell surface. Many of the microneme proteins identified so far contain adhesive domains. We here present the genomic and corresponding cDNA sequences coding for a 460-amino-acid (aa) microneme protein in Neospora caninum tachyzoites which, due to its homology to MIC1 in Toxoplasma gondii (TgMIC1), was named NcMIC1. The deduced NcMIC1 polypeptide sequence contains an N-terminal signal peptide of 20 aa followed by two tandemly internal repeats of 48 and 44 aa, respectively. Integrated into each repeat is a CXXXCG sequence motif reminiscent of the thrombospondin-related family of adhesive proteins. The positioning of this motif is strictly conserved in TgMIC1 and NcMIC1. The C-terminal part, comprised of 278 aa, was expressed in Escherichia coli, and antibodies affinity purified on recombinant NcMIC1 were used to confirm the localization within the micronemes by immunofluorescence and immunogold transmission electron microscopy of tachyzoites. Immunohistochemistry of mouse brains infected with tissue cysts showed that expression of this protein is reduced in the bradyzoite stage. Upon initiation of secretion by elevating the temperature to 37 degrees C, NcMIC1 is released into the medium supernatant. NcMIC1 binds to trypsinized, rounded Vero cells, as well as to Vero cell monolayers. Removal of glycosaminoglycans from the host cell surface and modulation of host cell surface glycosaminoglycan sulfation significantly reduces the binding of NcMIC1 to the host cell surface. Solid-phase binding assays employing defined glycosaminoglycans confirmed that NcMIC1 binds to sulfated glycosaminoglycans.     

基于IFA技术检测微线体蛋白2在艾美耳属球虫的空间分布

微线体蛋白2（Microneme protein2,Mic2）是艾美耳球虫入侵宿主细胞时分泌的主要功能蛋白之一。本研究中,以抗柔嫩艾美耳球虫微线体蛋白2（EtMic2）单克隆抗体为检测抗体,利用间接免疫荧光实验（IFA）检测了Mic2在柔嫩艾美耳球虫、和缓艾美耳球虫、斯氏艾美耳球虫和无残艾美耳球虫等不同种艾美耳属球虫的空间分布。结果显示,Mic2在不同种艾美耳属球虫中均定位于子孢子的顶部（微线体部位）,但表达强度存在差异。这提示Mic2可作为研究艾美耳属球虫相关蛋白空间分布的“参照物”。     

MIC6 associates with aldolase in host cell invasion by <Emphasis Type="Italic">Toxoplasma gondii</Emphasis>

The transmembrane microneme protein MIC6 and its partner MIC1, MIC4 comprise an adhesive complex that play important roles in host cell attachment by the obligate intracellular parasite Toxoplasma gondii. Successful penetration of host cells by T. gondii depends on coordinated interactions between MICs complex and the parasite's cytoskeleton. We have identified that the carboxy-terminal cytoplasmic domain (C domain) of MIC6 interacts with aldolase and the parasite cytoskeleton. Our finding uncovers new features regarding MIC6–aldolase interactions in host cell invasion by T. gondii.     

Targeting of soluble proteins to the rhoptries and micronemes in Toxoplasma gondii

Rhoptry and microneme organelles of the protozoan parasite Toxoplasma gondii are closely associated with host cell adhesion/invasion and establishment of the intracellular parasitophorous vacuole. In order to study the targeting of proteins to these specialized secretory organelles, we have engineered green fluorescent protein (GFP) fusions to the rhoptry protein ROP1 and the microneme protein MIC3. Both chimeras are correctly targeted to the appropriate organelles, permitting deletion analysis to map protein subdomains critical for targeting. The propeptide and a central 146 amino acid region of ROP1 are sufficient to target GFP to the rhoptries. More extensive deletions result in a loss of rhoptry targeting; the GFP reporter is diverted into the parasitophorous vacuole via dense granules. Certain MIC3 deletion mutants were also secreted into the parasitophorous vacuole via dense granules, supporting the view that this route constitutes the default pathway in T. gondii, and that specific signals are required for sorting to rhoptries and micronemes. Deletions within the cysteine-rich central region of MIC3 cause this protein to be arrested at various locations within the secretory pathway, presumably due to improper folding. Although correctly targeted to the appropriate organelles in living parasites, ROP1-GFP and MIC3-GFP fusion proteins were not secreted during invasion. GFP fusion proteins were readily secreted from dense granules, however, suggesting that protein secretion from rhoptries and micronemes might involve more than a simple release of organellar contents.     

Development and validation of real-time polymerase chain reaction assays specific to four species of Eimeria.

The development of quantitative real-time polymerase chain reaction (PCR) assays specific to Eimeria acervulina, Eimeria maxima, Eimeria necatrix and Eimeria tenella is described and validated. The PCR templates adopted include a fragment of a gene encoding a microneme protein and previously characterized species-specific random amplified polymorphic DNA (RAPD) sequences. The sensitivity of each assay allowed the consistent detection of between one and 10 parasite genomes, equivalent to between one or two sporulated oocysts or a fraction of a single mature schizont, unaffected by the presence of chicken (host) or other Eimeria species DNA. Regression coefficients in excess of 0.99 over linear ranges of at least six orders of magnitude, together with comparable PCR efficiencies, demonstrated the robust reproducibility of each assay and suggest that two or more may be successfully multiplexed. The species-specific assays described here, combined with a previously published generic Eimeria species real-time PCR, provide valuable components in a "tool box" to accurately quantify the presence of specific Eimeria species in environmental or within-host phases of the lifecycle with little specialist knowledge. The application of these assays may benefit chicken husbandry, veterinary practice, quality control of live vaccine production and scientific research.     

The Toxoplasma homolog of Plasmodium apical membrane antigen-1 (AMA-1) is a microneme protein secreted in response to elevated intracellular calcium levels

A monoclonal antibody (MAb) has been generated against a novel 63 kDa surface/apical antigen of Toxoplasma gondii tachyzoites which is identified here as TgAMA-1, the Toxoplasma homolog of Plasmodium apical membrane antigen-1 (AMA-1). Sequence analysis, phase partitioning in Triton X-114, and labeling of TgAMA-1 with iodonaphthalene azide all suggest that TgAMA-1 is a type I transmembrane protein. There is a high degree of sequence similarity between TgAMA-1 and Plasmodium AMA-1, most notably in the position of conserved cysteine residues within the protein's predicted extracellular domain. In contrast to full length Plasmodium AMA-1, which has previously been localized to the rhoptries, it is shown here by immunofluorescence and immunoelectron microscopy that intracellular TgAMA-1 is found in the micronemes. A 53 kDa N-terminal proteolytic fragment of TgAMA-1 is constitutively secreted from the parasite at 37 degrees C. As is the case with other microneme proteins, the proteolytic processing and secretion of TgAMA-1 is dramatically enhanced in response to treatments which increase intracellular calcium levels.     

Antibodies against thrombospondin-related anonymous protein do not inhibit Plasmodium sporozoite infectivity in vivo

Thrombospondin-related anonymous protein (TRAP), a candidate malaria vaccine antigen, is required for Plasmodium sporozoite gliding motility and cell invasion. For the first time, the ability of antibodies against TRAP to inhibit sporozoite infectivity in vivo is evaluated in detail. TRAP contains an A-domain, a well-characterized adhesive motif found in integrins. We modeled here a three-dimensional structure of the TRAP A-domain of Plasmodium yoelii and located regions surrounding the MIDAS (metal ion-dependent adhesion site), the presumed business end of the domain. Mice were immunized with constructs containing these A-domain regions but were not protected from sporozoite challenge. Furthermore, monoclonal and rabbit polyclonal antibodies against the A-domain, the conserved N terminus, and the repeat region of TRAP had no effect on the gliding motility or sporozoite infectivity to mice. TRAP is located in micronemes, secretory organelles of apicomplexan parasites. Accordingly, the antibodies tested here stained cytoplasmic TRAP brightly by immunofluorescence. However, very little TRAP could be detected on the surface of sporozoites. In contrast, a dramatic relocalization of TRAP onto the parasite surface occurred when sporozoites were treated with calcium ionophore. This likely mimics the release of TRAP from micronemes when a sporozoite contacts its target cell in vivo. Contact with hepatoma cells in culture also appeared to induce the release of TRAP onto the surface of sporozoites. If large amounts of TRAP are released in close proximity to its cellular receptor(s), effective competitive inhibition by antibodies may be difficult to achieve.     

Analysis of the human antibody response to thrombospondin-related anonymous protein of Plasmodium falciparum.

Thrombospondin-related anonymous protein (TRAP) of the malaria parasite Plasmodium falciparum shares two sequence motifs with other proteins which possess adhesive properties. Recently, findings indicate that TRAP is an antigen which contributes to antisporozoite immunity. We have cloned and expressed the TRAP coding sequences in Escherichia coli to investigate the human humoral immune response against this protein in a region of malaria endemicity of West Africa characterized by a seasonal transmission. Our results show that antibodies against TRAP are present in infected individuals. The anti-TRAP antibodies were analyzed in both a longitudinal and a prospective study. The longitudinal analysis shows seasonal fluctuations of the levels of specific antibodies as well as age-dependent quantitative differences. The immune response is long-lived in most of the adults and some of the older children but short-lived in young children. More importantly, the prospective analysis suggests that the presence of anti-TRAP antibodies in older children before the beginning of malaria transmission correlates with the subsequent control of parasite densities.     

Sequencing and analysis of chromosome 1 of Eimeria tenella reveals a unique segmental organization

Eimeria tenella is an intracellular protozoan parasite that infects the intestinal tracts of domestic fowl and causes coccidiosis, a serious and sometimes lethal enteritis. Eimeria falls in the same phylum (Apicomplexa) as several human and animal parasites such as Cryptosporidium, Toxoplasma, and the malaria parasite, Plasmodium. Here we report the sequencing and analysis of the first chromosome of E. tenella, a chromosome believed to carry loci associated with drug resistance and known to differ between virulent and attenuated strains of the parasite. The chromosome--which appears to be representative of the genome--is gene-dense and rich in simple-sequence repeats, many of which appear to give rise to repetitive amino acid tracts in the predicted proteins. Most striking is the segmentation of the chromosome into repeat-rich regions peppered with transposon-like elements and telomere-like repeats, alternating with repeat-free regions. Predicted genes differ in character between the two types of segment, and the repeat-rich regions appear to be associated with strain-to-strain variation.