Toxoplasma gondii: microneme protein MIC2

The phylum Apicomplexa contains parasites responsible for a variety of diseases including malaria, cryptosporidiosis, and toxoplasmosis. One of the common features of these parasites is that they contain a set of apical organelles whose sequential secretion is required for the invasion of host cells. Microneme proteins are the main adhesins involved in the attachment to the host cell surface by apicomplexans. The microneme protein MIC2, produced by Toxoplasma gondii, is conserved in apicomplexans and serves as a model to understand the first steps of invasion by the phylum. New data about the structure-function relationship of MIC2 reinforce the critical role of this protein in the successful invasion of cells by Toxoplasma and reveal potential therapeutic targets that may be used to control toxoplasmosis.     

Molecular signals in the trafficking of Toxoplasma gondii protein MIC3 to the micronemes

The protozoan parasite Toxoplasma gondii is equipped with a sophisticated secretory apparatus, including three distinct exocytic organelles, named micronemes, rhoptries, and dense granules. We have dissected the requirements for targeting the microneme protein MIC3, a key component of T. gondii infection. We have shown that MIC3 is processed in a post-Golgi compartment and that the MIC3 propeptide and epidermal growth factor (EGF) modules contain microneme-targeting information. The minimal requirement for microneme delivery is defined by the propeptide plus any one of the three EGF domains. We have demonstrated that the cleavage of the propeptide, the dimerization of MIC3, and the chitin binding-like sequence, which are crucial for host cell binding and virulence, are dispensable for proper targeting. Finally, we have shown that part of MIC3 is withheld in the secretory pathway in a cell cycle-dependent manner.     

The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin that binds to both the surface of the host cells and the surface of the parasite

Assay of the adhesion of cultured cells on Toxoplasma gondii tachyzoite protein Western blots identified a major adhesive protein, that migrated at 90 kDa in non-reducing gels. This band comigrated with the previously described microneme protein MIC3. Cellular binding on Western blots was abolished by MIC3-specific monoclonal and polyclonal antibodies. The MIC3 protein affinity purified from tachyzoite lysates bound to the surface of putative host cells. In addition, T. gondii tachyzoites also bound to immobilized MIC3. Immunofluorescence analysis of T. gondii tachyzoite invasion showed that MIC3 was exocytosed and relocalized to the surface of the parasite during invasion. The cDNA encoding MIC3 and the corresponding gene have been cloned, allowing the determination of the complete coding sequence. The MIC3 sequence has been confirmed by affinity purification of the native protein and N-terminal sequencing. The deduced protein sequence contains five partially overlapping EGF-like domains and a chitin binding-like domain, which can be involved in protein–protein or protein–carbohydrate interactions. Taken together, these results suggest that MIC3 is a new microneme adhesin of T. gondii .     

Toxoplasma gondii micronemal protein MIC1 is a lactose-binding lectin.

Host cell invasion by Toxoplasma gondii is a multistep process with one of the first steps being the apical release of micronemal proteins that interact with host receptors. We demonstrate here that micronemal protein 1 (MIC1) is a lactose-binding lectin. MIC1 and MIC4 were recovered in the lactose-eluted (Lac(+)) fraction on affinity chromatography on immobilized lactose of the soluble antigen fraction from tachyzoites of the virulent RH strain. MIC1 and MIC4 were both identified by N-terminal microsequencing. MIC4 was also identified by sequencing cDNA clones isolated from an expression library following screening with mouse polyclonal anti-60/70 kDa (Lac(+) proteins) serum. This antiserum localized the Lac(+) proteins on the apical region of T. gondii tachyzoites by confocal microscopy. The Lac(+) fraction induced hemagglutination (mainly type A human erythrocytes), which was inhibited by beta-galactosides (3 mM lactose and 12 mM galactose) but not by up to 100 mM melibiose (alpha-galactoside), fucose, mannose, or glucose or 0.2 mg/ml heparin. The lectin activity of the Lac(+) preparation was attributed to MIC1, because blotted MIC1, but not native MIC4, bound human erythrocyte type A and fetuin. The copurification of MIC1 and MIC4 may have been due to their association, as reported by others. These data suggest that MIC1 may act through its lectin activity during T. gondii infection.     

Complete resonance assignments for the MIC2 associated protein from Toxoplasma gondii

Toxoplasma gondii is an obligate parasite that infects most warm blood animals. Micronemal proteins actively involves in the invasion process, where TgMIC2 and TgM2AP complex plays vital roles. Complete NMR assignments for major fragment of TgM2AP were successfully obtained.     

Immunization with MIC1 and MIC4 induces protective immunity against Toxoplasma gondii

Host cell invasion by Toxoplasma gondii is tightly coupled to the apical release of micronemal proteins (MIC). In this work, we evaluated the protective effect encountered in C57BL/6 mice immunized with MIC1 and MIC4 purified from soluble tachyzoite antigens by affinity to immobilized lactose. The immunized mice presented high serum levels of IgG1 and IgG2b specific antibodies. MIC1/4-stimulated spleen cells from immunized mice produced IL-2, IL-12, IFN-gamma, IL-10, but not IL-4, suggesting the induction of a polarized Th1 type immune response. When orally challenged with 40 cysts of the ME49 strain, the immunized mice had 68% fewer brain cysts than the control mice. Immunization was associated with 80% survival of the mice challenged with 80 cysts, contrasting with 100% mortality of the non-immunized mice in the acute phase. In this phase, there was much lower parasitism in the lungs and small intestine of the immunized mice, and they did not exhibit the early-stage signs of intestinal necrosis, which was clearly detected in the control mice. Our data demonstrate that MIC1 and MIC4 triggered a protective response against toxoplasmosis, and that these antigens are targets for the further development of a vaccine.     

The microneme protein MIC4, or an MIC4-like protein, is expressed within the macrogamete and associated with oocyst wall formation in Toxoplasma gondii

The expression and localisation of MIC4, or an immuno-cross reacting MIC4-like protein, was examined in the enteric forms of Toxoplasma gondii using immunocytochemistry. In addition to being located within the micronemes of the merozoites, MIC4 or the MIC4-like protein was present within the macrogamete and was associated with the developing oocyst wall. The macrogamete is characterised by two types of structurally distinct wall forming bodies (WFB1 and 2). However, by immuno-electron microscopy, it was possible to identify two populations of dense granules (WFB1) which appear to form sequentially during macrogamete development. The first granules to form (WFB1a) stained positively with anti-MIC4 and were followed by MIC4 negative granules (WFB1b). During oocyst wall formation, the WFB1a and b sequentially released their contents onto the surface with WFB1a material forming an anti-MIC4 positive outer veil, while the WFB1b forms the electron dense outer layer of the oocyst wall. The inner layer was formed by WFB2. Thus, for the first time, it was possible to identify two populations of dense granules (WFB1a and b) involved in the formation of different parts of the oocyst wall. It was not possible to analyse the contents of macrogametes by western blot to unequivocally identify the antigen recognised by the polyclonal antisera as MIC4.     

Apicoplast targeting of a Toxoplasma gondii transmembrane protein requires a cytosolic tyrosine-based motif

Toxoplasma gondii, like most apicomplexan parasites, possesses an essential relict chloroplast, the apicoplast. Several apicoplast membrane proteins lack the bipartite targeting sequences of luminal proteins. Vesicles bearing these membrane proteins are detected during apicoplast enlargement, but the means of cargo selection remains obscure. We used a combination of deletion mutagenesis, point mutations and protein chimeras to identify a short motif prior to the first transmembrane domain of the T. gondii apicoplast phosphate transporter 1 (APT1) that is necessary for apicoplast trafficking. Tyrosine 16 was essential for proper localization; any substitution resulted in misdirection of APT1 to the Golgi body. Glycine 17 was also important, with significant Golgi body accumulation in the alanine mutant. Separation of at least eight amino acids from the transmembrane domain was required for full motif function. Similarly placed YG motifs are present in apicomplexan APT1 orthologs and the corresponding N‐terminal domain from Plasmodium vivax was able to route T. gondii APT1 to the apicoplast. Differential permeabilization showed that both the N‐ and C‐termini of APT1 are exposed to the cytosol. We propose that this YG motif facilitates APT1 trafficking via interactions that occur on the cytosolic face of nascent vesicles destined for the apicoplast.     

Transepithelial migration of Toxoplasma gondii involves an interaction of intercellular adhesion molecule 1 (ICAM-1) with the parasite adhesin MIC2

Toxoplasma gondii crosses non-permissive biological barriers such as the intestine, the blood-brain barrier and the placenta thereby gaining access to tissues where it most commonly causes severe pathology. Herein we show that in the process of migration Toxoplasma initially concentrates around intercellular junctions and probably uses a paracellular pathway to transmigrate across biological barriers. Parasite transmigration required viable and actively motile parasites. Interestingly, the integrity of host cell barriers was not altered during parasite transmigration. As intercellular adhesion molecule 1 (ICAM-1) is upregulated on cellular barriers during Toxoplasma infection, we investigated the role of this receptor in parasite transmigration. Soluble human ICAM-1 and ICAM-1 antibodies inhibited transmigration of parasites across cellular barriers implicating this receptor in the process of transmigration. Furthermore, human ICAM-1 immunoprecipitated the mature form of the parasite adhesin MIC2 present on the parasite surface, indicating that this interaction may contribute to cellular migration. These findings reveal that Toxoplasma exploits the natural cell trafficking pathways in the host to cross cellular barriers and disseminate to deep tissues.     

Identification and characterisation of Toxoplasma gondii protein farnesyltransferase

Prenylated proteins are involved in the regulation of DNA replication and cell cycling and have important roles in the regulation of cell proliferation. Protein farnesyltransferase and protein geranylgeranyltransferase are the two enzymes responsible for catalysing isoprene lipid modifications. Recently these enzymes have been targets for the development of cancer chemotherapeutics. Using metabolic labelling we identified isoprenylated proteins which suggests the presence of protein farnesyltransferase in Toxoplasma gondii. T. gondii protein farnesyltransferase is heat-labile and requires Mg(2+) and Zn(2+) ions for full activity. Peptidomimetic analogues as well as short synthetic peptides were tested in vitro as possible competitors for farnesyltransferase substrates. We found that the synthetic peptide (KTSCVIA) specifically inhibited T. gondiiprotein farnesyltransferase but not mammalian (HeLa cells) farnesyltransferase. Therefore this study suggests the possible development of specific inhibitors of T. gondiiprotein farnesyltransferase as an approach to parasitic protozoa therapy.     

Rapid invasion of host cells by Toxoplasma requires secretion of the MIC2-M2AP adhesive protein complex

Vertebrate cells are highly susceptible to infection by obligate intracellular parasites such as Toxoplasma gondii, yet the mechanism by which these microbes breach the confines of their target cell is poorly understood. While it is thought that Toxoplasma actively invades by secreting adhesive proteins from internal organelles called micronemes, no genetic evidence is available to support this contention. Here, we report successful disruption of M2AP, a microneme protein tightly associated with an adhesive protein called MIC2. M2AP knockout parasites were >80% impaired in host cell entry. This invasion defect was likely due to defective expression of MIC2, which partially accumulated in the parasite endoplasmic reticulum and Golgi. M2AP knockout parasites were also unable to rapidly secrete MIC2, an event that normally accompanies parasite attachment to a target cell. These findings indicate a critical role for the MIC2-M2AP protein complex in parasite invasion.     

Detailed insights from microarray and crystallographic studies into carbohydrate recognition by microneme protein 1 (MIC1) of Toxoplasma gondii

The intracellular protozoan Toxoplasma gondii is among the most widespread parasites. The broad host cell range of the parasite can be explained by carbohydrate microarray screening analyses that have demonstrated the ability of the T. gondii adhesive protein, TgMIC1, to bind to a wide spectrum of sialyl oligosaccharide ligands. Here, we investigate by further microarray analyses in a dose-response format the differential binding of TgMIC1 to 2-3- and 2-6-linked sialyl carbohydrates. Interestingly, two novel synthetic fluorinated analogs of 3′SiaLacNAc_1–4 and 3′SiaLacNAc_1–3 were identified as highly potent ligands. To understand the structural basis of the carbohydrate binding specificity of TgMIC1, we have determined the crystal structures of TgMIC1 micronemal adhesive repeat (MAR)-region (TgMIC1-MARR) in complex with five sialyl-N-acetyllactosamine analogs. These crystal structures have revealed a specific, water-mediated hydrogen bond network that accounts for the preferential binding of TgMIC1-MARR to arrayed 2-3-linked sialyl oligosaccharides and the high potency of the fluorinated analogs. Furthermore, we provide strong evidence for the first observation of a C—F···H—O hydrogen bond within a lectin-carbohydrate complex. Finally, detailed comparison with other oligosaccharide-protein complexes in the Protein Data Bank (PDB) reveals a new family of sialic-acid binding sites from lectins in parasites, bacteria, and viruses.     

Plasmodial ortholog of Toxoplasma gondii rhoptry neck protein 3 is localized to the rhoptry body

The proteins in apical organelles of Plasmodium falciparum merozoite play an important role in invasion into erythrocytes. Several rhoptry neck (RON) proteins have been identified in rhoptry proteome of the closely-related apicomplexan parasite, Toxoplasma gondii. Recently, three of P. falciparum proteins orthologous to TgRON proteins, PfRON2, 4 and 5, were found to be located in the rhoptry neck and interact with the micronemal protein apical membrane antigen 1 (PfAMA1) to form a moving junction complex that helps the invasion of merozoite into erythrocyte. However, the other P. falciparum RON proteins have yet to be characterized. Here, we determined that "PFL2505c" (hereafter referred to as pfron3) is the ortholog of the tgron3 in P. falciparum and characterized its protein expression profile, subcellular localization, and complex formation. Protein expression analysis revealed that PfRON3 was expressed primarily in late schizont stage parasites. Immunofluorescence microscopy (IFA) showed that PfRON3 localizes in the apical region of P. falciparum merozoites. Results from immunoelectron microscopy, along with IFA, clarified that PfRON3 localizes in the rhoptry body and not in the rhoptry neck. Even after erythrocyte invasion, PfRON3 was still detectable at the parasite ring stage in the parasitophorous vacuole. Moreover, co-immunoprecipitation studies indicated that PfRON3 interacts with PfRON2 and PfRON4, but not with PfAMA1. These results suggest that PfRON3 partakes in the novel PfRON complex formation (PfRON2, 3, and 4), but not in the moving junction complex (PfRON2, 4, 5, and PfAMA1). The novel PfRON complex, as well as the moving junction complex, might play a fundamental role in erythrocyte invasion by merozoite stage parasites.     

Sequence Diversity in MIC6 Gene among Toxoplasma gondii Isolates from Different Hosts and Geographical Locations

Toxoplasma gondii is an opportunistic protozoan parasite that can infect almost all warm-blooded animals including humans with a worldwide distribution. Micronemes play an important role in invasion process of T. gondii, associated with the attachment, motility, and host cell recognition. In this research, sequence diversity in microneme protein 6 (MIC6) gene among 16 T. gondii isolates from different hosts and geographical regions and 1 reference strain was examined. The results showed that the sequence of all the examined T. gondii strains was 1,050 bp in length, and their A + T content was between 45.7% and 46.1%. Sequence analysis presented 33 nucleotide mutation positions (0-1.1%), resulting in 23 amino acid substitutions (0-2.3%) aligned with T. gondii RH strain. Moreover, T. gondii strains representing the 3 classical genotypes (Type I, II, and III) were separated into different clusters based on the locus of MIC6 using phylogenetic analyses by Bayesian inference (BI), maximum parsimony (MP), and maximum likelihood (ML), but T. gondii strains belonging to ToxoDB #9 were separated into different clusters. Our results suggested that MIC6 gene is not a suitable marker for T. gondii population genetic studies.     

The toxoplasma micronemal protein MIC4 is an adhesin composed of six conserved apple domains

The initial stage of invasion by apicomplexan parasites involves the exocytosis of the micronemes-containing molecules that contribute to host cell attachment and penetration. MIC4 was previously described as a protein secreted by Toxoplasma gondii tachyzoites upon stimulation of micronemes exocytosis. We have microsequenced the mature protein, purified after discharge from micronemes and cloned the corresponding gene. The deduced amino acid sequence of MIC4 predicts a 61-kDa protein that contains 6 conserved apple domains. Apple domains are composed of six spacely conserved cysteine residues which form disulfide bridges and are also present in micronemal proteins from two closely related apicomplexan parasites, Sarcocystis muris and Eimeria species, and several mammalian serum proteins, including kallikrein. Here we show that MIC4 localizes in the micronemes of all the invasive forms of T. gondii, tachyzoites, bradyzoites, sporozoites, and merozoites. The protein is proteolytically processed both at the N and the C terminus only upon release from the organelle. MIC4 binds efficiently to host cells, and the adhesive motif maps in the most C-terminal apple domain.     

The immunobiology of the innate response to Toxoplasma gondii

Toxoplasma gondii is a unique intracellular parasite. It can infect a variety of cells in virtually all warm-blooded animals. It has a worldwide distribution and, overall, around one-third of people are seropositive for the parasite, with essentially the entire human population being at risk of infection. For most people, T. gondii causes asymptomatic infection but the parasite can cause serious disease in the immunocompromised and, if contracted for the first time during pregnancy, can cause spontaneous abortion or congenital defects, which have a substantial emotional, social and economic impact. Toxoplasma gondii provokes one of the most potent innate, pro-inflammatory responses of all infectious disease agents. It is also a supreme manipulator of the immune response so that innate immunity to T. gondii is a delicate balance between the parasite and its host involving a coordinated series of cellular interactions involving enterocytes, neutrophils, dendritic cells, macrophages and natural killer cells. Underpinning these interactions is the regulation of complex molecular reactions involving Toll-like receptors, activation of signalling pathways, cytokine production and activation of anti-microbial effector mechanisms including generation of reactive nitrogen and oxygen intermediates.     

Converting and hoarding driven by protein phosphorylation in Toxoplasma gondii

Successful parasitism relies on the evasion of adversarial host responses. Wang et al. have recently shown that Toxoplasma gondii relies on the protein phosphatase 2A (PP2A) to cause persisting infections. The phosphatase controls the development of dormant parasite stages and the accumulation of sugar supplies.     

The role of host-derived heat-shock protein in immunity against Toxoplasma gondii infection

Heat-shock proteins (HSPs) are evolutionarily highly conserved polypeptides synthesized by cells to preserve cellular functions under a variety of stressful conditions, including infections. In infections, both host cells and pathogens express HSPs, although the role of these molecules in the host-pathogen relationship is elusive. Here, Hajime Hisaeda and Kunisuke Himeno show that a correlation exists between the 65 kDa HSP molecule (HSP65) and protection against Toxoplasma gondii infection, suggesting that this protein contributes to the host defense system. These findings may help in the understanding of the complicated host-parasite relationship.     

Complete NMR assignments for the second EGF domain of MIC6 from Toxoplasma gondii and re-assignment in complex with the galectin-like domain of MIC1

Toxoplasma gondii is the causative agent of toxoplasmosis. Here we present a complete set of NMR assignments for the second EGF domain from microneme protein 6 and its re-assignment in complex with the galectin-like domain from microneme protein 1.