Neospora caninum Microneme Protein NcMIC3: Secretion, Subcellular Localization, and Functional Involvement in Host Cell Interaction

In apicomplexan parasites, host cell adhesion and subsequent invasion involve the sequential release of molecules originating from secretory organelles named micronemes, rhoptries, and dense granules. Microneme proteins have been shown to be released at the onset of the initial contact between the parasite and the host cell and thus mediate and establish the physical interaction between the parasite and the host cell surface. This interaction most likely involves adhesive domains found within the polypeptide sequences of most microneme proteins identified to date. NcMIC3 is a microneme-associated protein found in Neospora caninum tachyzoites and bradyzoites, and a large portion of this protein is comprised of a stretch of four consecutive epidermal growth factor (EGF)-like domains. We determined the subcellular localization of NcMIC3 prior to and following host cell invasion and found that NcMIC3 was secreted onto the tachyzoite surface immediately following host cell lysis in a temperature-dependent manner. Surface-exposed NcMIC3 could be detected up to 2 to 3 h following host cell invasion, and at later time points the distribution of the protein was again restricted to the micronemes. In vitro secretion assays using purified tachyzoites showed that following secretion onto the surface, NcMIC3 was largely translocated towards the posterior end of the parasite, employing a mechanism which requires a functional actin microfilament system. Following this, the protein remained bound to the parasite surface, since it could not be detected in a soluble form in respective culture supernatants. Secretion of NcMIC3 onto the surface resulted in an outward exposure of the EGF-like domains and coincided with an increased capacity of N. caninum tachyzoites to adhere to Vero cell monolayers in vitro, a capacity which could be inhibited by addition of antibodies directed against the EGF-like domains. NcMIC3 is a prominent component of Triton X-100 lysates of tachyzoites, and cosedimentation assays employing prefixed Vero cells showed that the protein binds to the Vero cell surface. In addition, the EGF-like domains, expressed as recombinant proteins in Escherichia coli, also interacted with the Vero cell surface, while binding of NcSRS2 and NcSAG1, the major immunodominant surface antigens, was not as efficient. Our data are indicative of a functional role of NcMIC3 in host cell infection.     

Identification and characterization of a Neospora caninum microneme-associated protein (NcMIC4) that exhibits unique lactose-binding properties

Microneme proteins have been shown to play an important role in the early phase of host cell adhesion, by mediating the contact between the parasite and host cell surface receptors. In this study we have identified and characterized a lectin-like protein of Neospora caninum tachyzoites which was purified by alpha-lactose-agarose affinity chromatography. Upon separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this lactose-binding protein migrated at 70 and 55 kDa under reducing and nonreducing conditions, respectively. Immunofluorescence and immunogold electron microscopy with affinity-purified antibodies showed that the protein was associated with the tachyzoite micronemes. Mass spectrometry analyses and expressed sequence tag database mining revealed that this protein is a member of the Neospora microneme protein family; the protein was named NcMIC4 (N. caninum microneme protein 4). Upon two-dimensional gel electrophoresis, NcMIC4 separated into seven distinct isoforms. Incubation of extracellular parasites at 37 degrees C resulted in the secretion of NcMIC4 into the medium as a soluble protein, and the secreted protein exhibited a slightly reduced M(r) but retained its lactose-binding properties. Immunofluorescence was used to investigate the temporal and spatial distribution of NcMIC4 in tachyzoites entering their host cells and showed that reexpression of NcMIC4 took place 30 min after entry into the host cell. Incubation of secreted fractions and purified NcMIC4 with Vero cells demonstrated binding of NcMIC4 to Vero cells as well as binding to chondroitin sulfate A glycosaminoglycans.     

Molecular characterization of a thrombospondin-related anonymous protein homologue in Neospora caninum

Thrombospondin-related anonymous protein (TRAP) family members participate in attachment and invasion of host cells by apicomplexan parasites. A TRAP homologue in Neospora caninum strain Nc-1 (NcMIC2) was cloned, sequenced and found to be 61% identical (75% similar) at the amino acid level to Toxoplasma gondii MIC2 (TgMIC2). Similar to TgMIC2, the predicted amino acid sequence of NcMIC2 contains one integrin-like domain (I or A domain), five thrombospondin (TSP) repeats, a putative transmembrane spanning region and intracellular C-terminus, and was localized to micronemes by cryo-immunoelectron microscopy. The secretion of NcMIC2 was temperature dependent and was induced at or above 25 degrees C. The secreted form of NcMIC2 released into the medium was found to be proteolytically processed such that it lacked the C-terminal domain. Secretion of NcMIC2 was regulated by calcium, since several agents which raise intracellular calcium levels were shown to promote NcMIC2 secretion and chelation of [Ca(2+)](i) abrogated release. As a member of the growing family of apicomplexan TRAP proteins, NcMIC2 may play an important role in attachment and invasion by N. caninum into host cells.     

Molecular characterization of TgMIC5, a proteolytically processed antigen secreted from the micronemes of Toxoplasma gondii

During invasion of host cells, Toxoplasma gondii discharges the contents of small, apically located secretory organelles called micronemes. Micronemal proteins are known to be necessary for both parasite motility and invasion of host cells. To further define the contents of Toxoplasma micronemes, we used cell fractionation and secretion-modulating drugs to identify six novel, putative micronemal proteins. In this paper we describe preliminary characterization of one of these novel proteins, TgMIC5. Molecular cloning and DNA sequence analysis of the TgMIC5 cDNA and gene revealed that it encodes a previously identified immunodominant antigen called H4. TgMIC5 also possesses a consensus sequence unique to members of the parvulin family of peptidyl-prolyl cis-trans isomerases (PPIases). TgMIC5 is expressed as a preproprotein, which is proteolytically processed to a proprotein by signal peptidase before being further processed to a mature protein of 22 kDa. Using a combination of protein secretion experiments, immunofluorescence and immunoelectron microscopy, we demonstrated that TgMIC2 is stored in the micronemes of T. gondii tachyzoites before it is secreted into the surrounding medium. Based on its homology with parvulin-like PPIases, TgMIC5 may assist in the folding of other micronemal proteins that function in invasion of host cells by T. gondii tachyzoites.     

A microneme protein from Eimeria tenella with homology to the Apple domains of coagulation factor XI and plasma pre-kallikrein

Microneme organelles are present in all apicomplexan protozoa and contain proteins that are critical for parasite motility and host cell invasion. One apicomplexan-wide family of microneme proteins has been identified with members that are characterised by the possession of thrombospondin type I repeats, conserved adhesive motifs which are implicated in binding to glycosaminoglycan chains. In this paper we describe a micronemal glycoprotein, EtMIC 5, from Eimeria tenella which contains eleven cysteine-rich motifs that have striking similarity to the adhesive Apple (A-) domains of blood coagulation factor XI and plasma pre-kallikrein. EtMIC 5 is confined to an intracellular location in resting sporozoites but is translocated to the parasite surface and secreted into the culture supernatant during parasite infection of MDBK cells. During intracellular replication, the protein is switched off in early schizogony and is then re-expressed within the apical tips of newly formed merozoites. A-domain sequences were also found in microneme proteins from Sarcocystis muris and Toxoplasma gondii and in a protein of unknown localisation from Eimeria acervulina. These studies suggest that A-domain containing proteins may comprise a novel apicomplexan-wide family of microneme adhesins.     

Attachment ligands of viable Toxoplasma gondii induce soluble immunosuppressive factors in human monocytes

Previous studies have demonstrated that surface antigen proteins, in particular SAG-1, of Toxoplasma gondii are important to this parasite as attachment ligands for the host cell. An in vitro assay was developed to test whether these ligands and other secretory proteins are involved in the immune response of human cells to toxoplasma. Human monocytes were infected with tachyzoites in the presence of antiparasite antibodies, and their effect on mitogen-induced lymphoproliferation was examined. The presence of antibody to either parasite-excreted proteins (MIC-1 and MIC-2) or surface proteins (SAG-1 and SAG-2) during infection neutralized the marked decrease seen in mitogen-induced lymphoproliferation in the presence of infected monocytes. Conversely, antibodies to other secreted proteins (ROP-1) and cytoplasmic molecules had no effect on parasite-induced, monocyte-mediated downregulation. Fluorescence microscope analysis detected microneme and surface antigen proteins on the monocyte cell surface during infection. These results suggest that microneme and surface antigen proteins trigger monocytes to downregulate mitogen-induced lymphoproliferation.     

In vitro induction of Neospora caninum bradyzoites in vero cells reveals differential antigen expression, localization, and host-cell recognition of tachyzoites and bradyzoites

We report on an optimized method for the in vitro culture of tissue cyst-forming Neospora caninum bradyzoites in Vero cells and the separation of viable parasites from host cells. Treatment of tachyzoite-infected Vero cell cultures with 17 microM sodium nitroprusside for 8 days severely scaled down parasite proliferation, led to reduced expression of tachyzoite surface antigens, and induced the expression of the bradyzoite marker NcBAG1 and the cyst wall antigen recognized by the monoclonal antibody MAbCC2. Transmission electron microscopy demonstrated that intracellular parasites were located within parasitophorous vacuoles that were surrounded by a cyst wall-like structure, and the dense granule antigens NcGRA1, NcGRA2, and NcGRA7 were incorporated into the cyst wall. Adhesion-invasion assays employing purified tachyzoites and bradyzoites showed that tachyzoites adhered to, and invaded, Vero cells with higher efficiency than bradyzoites. However, removal of terminal sialic acid residues from either the host cell or the parasite surface increased the invasion of Vero cells by bradyzoites, but not tachyzoites.     

Subcellular localization and functional characterization of Nc-p43, a major Neospora caninum tachyzoite surface protein.

Neospora caninum is a recently identified coccidian parasite which shares many features with, but is clearly distinct from, Toxoplasma gondii. N. caninum tachyzoites infect a wide range of mammalian cells both in vivo and in vitro. The mechanisms by which infection is achieved are largely unknown. Recent evidence has suggested that a receptor-ligand system in which one or several host cell receptors bind to one or several parasite ligands is involved. Parasite cell surface-associated molecules such as the recently identified Nc-p43 antigen are prime suspects for being implicated in this physical interaction. In this study it is shown that invasion of Vero cell monolayers by N. caninum tachyzoites in vitro is impaired on incubation of parasites with subagglutinating amounts of affinity-purified antibodies directed against Nc-p43. Postembedding immunogold labeling with anti-Nc-p43 antibodies demonstrated that Nc-p43 is localized not only on the parasite cell surface but also within dense granules and rhoptries. The fate of Nc-p43 during intracellular proliferation of N. caninum tachyzoites and subsequent maturation of the parasitophorous vacuole was also studied.     

Moraxella catarrhalis binding to host cellular receptors is mediated by sequence-specific determinants not conserved among all UspA1 protein variants

The Moraxella catarrhalis ubiquitous surface proteins (UspAs) are autotransporter molecules reported to interact with a variety of different host proteins and to affect processes ranging from serum resistance to cellular adhesion. The role of UspA1 as an adhesin has been confirmed with a number of different human cell types and is mediated by binding to eukaryotic proteins including carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs), fibronectin, and laminin. A distinct difference in the ability of prototypical M. catarrhalis strains to adhere to CEACAM-expressing cell lines prompted us to perform strain-specific structure-function analyses of UspA1 proteins. In this study, we characterized CEACAM binding by a diverse set of UspA1 proteins and showed that 3 out of 10 UspA1 proteins were incapable of binding CEACAM. This difference resulted from the absence of a distinct CEACAM binding motif in nonadhering strains. Our sequence analysis also revealed a single M. catarrhalis isolate that lacked the fibronectin-binding motif and was defective in adherence to Chang conjunctival epithelial cells. These results clearly demonstrate that UspA1-associated adhesive functions are not universally conserved. Instead, UspA1 proteins must be considered as variants with the potential to confer both different cell tropisms and host cell responses.     

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.     

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.     

Cell adhesion molecules in invertebrate immunity.

Cell adhesion is essential in immunity in invertebrates, e.g., in the cellular immune responses of encapsulation and nodule formation. Here cell adhesion molecules shown or suggested to be involved in invertebrate immunity are reviewed. Blood cells of the crayfish, Pacifastacus leniusculus, can release a cell-adhesive and opsonic peroxidase, peroxinectin. A site containing the motif, KGD, appears to be adhesive by binding to a transmembrane receptor of the integrin family on the blood cells. Peroxinectin also binds a peripheral blood cell surface CuZn-superoxide dismutase. The peroxidase-integrin interaction appears to have evolved early and seems conserved; human myeloperoxidase supports cell adhesion via the alphaMbeta2 integrin. There is evidence for peroxinectin-like proteins in other arthropods. Effects by RGD peptides indicate that integrins mediate blood cell adhesion and cellular immunity in diverse invertebrate species. Other invertebrate blood cell molecules proposed to be involved in adhesion include the insect plasmatocyte-spreading peptide, as well as soluble and transmembrane proteins which show some similarity to vertebrate adhesive or extracellular matrix molecules. Proteins such as the Ig family member hemolin, or proteins found in insects that are hosts for parasitic wasps, inhibit cell adhesion and may regulate or block cellular immunity.     

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.     

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.     

Gliding motility leads to active cellular invasion by Cryptosporidium parvum sporozoites

We examined gliding motility and cell invasion by an early-branching apicomplexan, Cryptosporidium parvum, which causes diarrheal disease in humans and animals. Real-time video microscopy demonstrated that C. parvum sporozoites undergo circular and helical gliding, two of the three stereotypical movements exhibited by Toxoplasma gondii tachyzoites. C. parvum sporozoites moved more rapidly than T. gondii sporozoites, which showed the same rates of motility as tachyzoites. Motility by C. parvum sporozoites was prevented by latrunculin B and cytochalasin D, drugs that depolymerize the parasite actin cytoskeleton, and by the myosin inhibitor 2,3-butanedione monoxime. Imaging of the initial events in cell entry by Cryptosporidium revealed that invasion occurs rapidly; however, the parasite does not enter deep into the cytosol but rather remains at the cell surface in a membrane-bound compartment. Invasion did not stimulate rearrangement of the host cell cytoskeleton and was inhibited by cytochalasin D, even in host cells that were resistant to the drug. Our studies demonstrate that C. parvum relies on a conserved actin-myosin motor for motility and active penetration of its host cell, thus establishing that this is a widely conserved feature of the Apicomplexa.     

The cysteine-rich regions of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> RON2 bind with host erythrocyte and AMA1 during merozoite invasion

Invasion of Plasmodium falciparum merozoites into host erythrocyte involves a series of highly specific and sequential interaction between merozoite and host erythrocyte surface protein. The key step in the invasion process is the formation of a tight protein–protein interaction between host and parasite called as moving junction. A number of parasite proteins secreted from two organelles, microneme and rhoptry, play a role in initial interaction and junction formation between merozoite with host red blood cells (RBCs) during the invasion process. In the present study, we investigated the role of different domains of a P. falciparum rhoptry neck protein PfRON2. Immunofluorescence assay revealed close association of PfAMA1 and PfRON2 in the merozoites during the invasion process. PfRON2 domains were expressed on COS-7 cell surface, and their interaction was analysed with host RBCs and PfAMA1 protein by rosetting assays. The rosetting assays suggest that the C-terminal cysteine-rich domain of PfRON2 plays a role in binding with host erythrocyte. The C-terminal as well as the central cysteine-rich domain of PfRON2 interact with PfAMA1; this binding can be inhibited by monoclonal antibody (mAb 4 G2) against PfAMA1, suggesting that the hydrophobic groove of PfAMA1 binds to PfRON2. These results suggest that PfRON2 plays a role in merozoite invasion and thus it can be an important vaccine candidate antigen.     

Toxoplasma gondii homologue of plasmodium apical membrane antigen 1 is involved in invasion of host cells

Proteins with constitutive or transient localization on the surface of Apicomplexa parasites are of particular interest for their potential role in the invasion of host cells. We describe the identification and characterization of TgAMA1, the Toxoplasma gondii homolog of the Plasmodium apical membrane antigen 1 (AMA1), which has been shown to elicit a protective immune response against merozoites dependent on the correct pairing of its numerous disulfide bonds. TgAMA1 shows between 19% (Plasmodium berghei) and 26% (Plasmodium yoelii) overall identity to the different Plasmodium AMA1 homologs and has a conserved arrangement of 16 cysteine residues and a putative transmembrane domain, indicating a similar architecture. The single-copy TgAMA1 gene is interrupted by seven introns and is transcribed into an mRNA of approximately 3.3 kb. The TgAMA1 protein is produced during intracellular tachyzoite replication and initially localizes to the micronemes, as determined by immunofluorescence assay and immunoelectron microscopy. Upon release of mature tachyzoites, TgAMA1 is found distributed predominantly on the apical end of the parasite surface. A approximately 54-kDa cleavage product of the large ectodomain is continuously released into the medium by extracellular parasites. Mouse antiserum against recombinant TgAMA1 blocked invasion of new host cells by approximately 40%. This and our inability to produce a viable TgAMA1 knock-out mutant indicate that this phylogenetically conserved protein fulfills a key function in the invasion of host cells by extracellular T. gondii tachyzoites.