Galactose recognition by the apicomplexan parasite Toxoplasma gondii

Toxosplasma gondii is the model parasite of the phylum Apicomplexa, which contains numerous obligate intracellular parasites of medical and veterinary importance, including Eimeria, Sarcocystis, Cryptosporidium, Cyclospora, and Plasmodium species. Members of this phylum actively enter host cells by a multistep process with the help of microneme protein (MIC) complexes that play important roles in motility, host cell attachment, moving junction formation, and invasion. T. gondii (Tg)MIC1-4-6 complex is the most extensively investigated microneme complex, which contributes to host cell recognition and attachment via the action of TgMIC1, a sialic acid-binding adhesin. Here, we report the structure of TgMIC4 and reveal its carbohydrate-binding specificity to a variety of galactose-containing carbohydrate ligands. The lectin is composed of six apple domains in which the fifth domain displays a potent galactose-binding activity, and which is cleaved from the complex during parasite invasion. We propose that galactose recognition by TgMIC4 may compromise host protection from galectin-mediated activation of the host immune system.     

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.     

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.     

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.     

Embryo vaccination against Eimeria tenella and E. acervulina infections using recombinant proteins and cytokine adjuvants

Avian coccidiosis is an intestinal disease caused by protozoa of the genus Eimeria. To investigate the potential of recombinant protein vaccines to control coccidiosis, we cloned 2 Eimeria sp. genes (EtMIC2 and 3-1E), expressed and purified their encoded proteins, and determined the efficacy of in ovo immunization to protect against Eimeria infections. Immunogen-specific serum antibody titers, parasite fecal shedding, and body weight gains were measured as parameters of disease. When administered alone, the recombinant EtMIC2 gene product induced significantly higher antibody responses, lower oocyst fecal shedding, and increased weight gains compared with nonvaccinated controls following infection with E. tenella. Combined embryo immunization with the EtMIC2 protein plus chicken cytokine or chemokine genes demonstrated that all 3 parameters of vaccination were improved compared with those of EtMIC2 alone. In particular, covaccination with EtMIC2 plus interleukin (IL)-8, IL-16, transforming growth factor-beta4, or lymphotactin significantly decreased oocyst shedding and improved weight gains beyond those achieved by EtMIC2 alone. Finally, individual vaccination with either EtMIC2 or 3-1E stimulated protection against infection by the heterologous parasite E. acervulina. Taken together, these results indicate that in ovo vaccination with the EtMIC2 protein plus cytokine/chemokine genes may be an effective method to control coccidiosis.     

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.     

Atomic resolution insight into host cell recognition by Toxoplasma gondii

The obligate intracellular parasite Toxoplasma gondii, a member of the phylum Apicomplexa that includes Plasmodium spp., is one of the most widespread parasites and the causative agent of toxoplasmosis. Micronemal proteins (MICs) are released onto the parasite surface just before invasion of host cells and play important roles in host cell recognition, attachment and penetration. Here, we report the atomic structure for a key MIC, TgMIC1, and reveal a novel cell-binding motif called the microneme adhesive repeat (MAR). Using glycoarray analyses, we identified a novel interaction with sialylated oligosaccharides that resolves several prevailing misconceptions concerning TgMIC1. Structural studies of various complexes between TgMIC1 and sialylated oligosaccharides provide high-resolution insights into the recognition of sialylated oligosaccharides by a parasite surface protein. We observe that MAR domains exist in tandem repeats, which provide a highly specialized structure for glycan discrimination. Our work uncovers new features of parasite-receptor interactions at the early stages of host cell invasion, which will assist the design of new therapeutic strategies.     

Solution structure of a PAN module from the apicomplexan parasite Eimeria tenella

Micronemes, specialised organelles found in all apicomplexan parasites, secrete molecules that are essential for parasite attachment and invasion of host cells. EtMIC5 is one such microneme protein that contains eleven tandemly repeating modules. These modules have homology with the PAN module superfamily. Members of this family are found in blood clotting proteins, some growth factors and some nematode proteins. This paper presents the structure of the 9^th PAN module in EtMIC5, determined using high resolution NMR. The structure shows similarities to and some differences from the N-terminal module of hepatocyte growth factor (HGF), the only previous member of the PAN family with known structure. AbbreviationsNMR – nuclear magnetic resonance; NOE – nuclear Overhauser enhancement; NOESY – NOE spectroscopy; COSY – correlated spectroscopy; TOCSY – total correlated spectroscopy; HSQC – hetero nuclear single quantum coherence; HMQC-J – hetero nuclear multiple quantum coherence-J coupling; MICs – microneme proteins; EtMIC5 – a microneme protein from Eimeria tenella; Apple9 – the ninth Apple repeat of EtMIC5; FXI – blood coagulation factor XI; PK – plasma prekallikrein; HGF – hepatocyte growth factor.     

Trans-genera reconstitution and complementation of an adhesion complex in Toxoplasma gondii

Eimeria tenella and Toxoplasma gondii are obligate intracellular parasites belonging to the phylum Apicomplexa. In T. gondii, the microneme protein TgMIC2 contains two well-defined adhesive motifs and is thought to be a key participant in the attachment and invasion of host cells. However, several attempts by different laboratories to generate a knockout (KO) of TgMIC2 have failed, implying that TgMIC2 is an essential gene. As Eimeria and Toxoplasma utilize the same mechanisms of invasion and have highly conserved adhesive proteins, we hypothesized that the orthologous molecule in Eimeria, EtMIC1, could functionally substitute in Toxoplasma to allow a knockout of TgMIC2. TgMIC2 is partnered with a protein called TgM2AP, which corresponds to EtMIC2 in Eimeria. Because the activity of TgMIC2 is most likely tightly linked to its association with TgM2AP, it was thought that the activity of EtMIC1 might similarly require its partner EtMIC2. EtMIC1 and EtMIC2 were introduced into T. gondii, and the presence of EtMIC1 allowed the first knockout clone of TgMIC2 to be obtained. The TgMIC2 KO resulted in significantly decreased numbers of invaded parasites compared to the parental clone. In the absence of TgMIC2, TgM2AP was incorrectly processed and mistargeted to the parasitophorous vacuole instead of the micronemes. These findings indicate that the EtMIC1 can compensate for the essential requirement of TgMIC2, but it cannot fully functionally substitute for TgMIC2 in the invasion process or for supporting the correct maturation and targeting of TgM2AP.     

Members of a Novel Protein Family Containing Microneme Adhesive Repeat Domains Act as Sialic Acid-binding Lectins during Host Cell Invasion by Apicomplexan Parasites

Numerous intracellular pathogens exploit cell surface glycoconjugates for host cell recognition and entry. Unlike bacteria and viruses, Toxoplasma gondii and other parasites of the phylum Apicomplexa actively invade host cells, and this process critically depends on adhesins (microneme proteins) released onto the parasite surface from intracellular organelles called micronemes (MIC). The microneme adhesive repeat (MAR) domain of T. gondii MIC1 (TgMIC1) recognizes sialic acid (Sia), a key determinant on the host cell surface for invasion by this pathogen. By complementation and invasion assays, we demonstrate that TgMIC1 is one important player in Sia-dependent invasion and that another novel Sia-binding lectin, designated TgMIC13, is also involved. Using BLAST searches, we identify a family of MAR-containing proteins in enteroparasitic coccidians, a subclass of apicomplexans, including T. gondii, suggesting that all these parasites exploit sialylated glycoconjugates on host cells as determinants for enteric invasion. Furthermore, this protein family might provide a basis for the broad host cell range observed for coccidians that form tissue cysts during chronic infection. Carbohydrate microarray analyses, corroborated by structural considerations, show that TgMIC13, TgMIC1, and its homologue Neospora caninum MIC1 (NcMIC1) share a preference for α2–3- over α2–6-linked sialyl-N-acetyllactosamine sequences. However, the three lectins also display differences in binding preferences. Intense binding of TgMIC13 to α2–9-linked disialyl sequence reported on embryonal cells and relatively strong binding to 4-O-acetylated-Sia found on gut epithelium and binding of NcMIC1 to 6′sulfo-sialyl Lewis^x might have implications for tissue tropism.     

Forward targeting of Toxoplasma gondii proproteins to the micronemes involves conserved aliphatic amino acids

Like other apicomplexan parasites, Toxoplasma gondii actively invades host cells using a combination of secretory proteins and an acto-myosin motor system. Micronemes are the first set of proteins secreted during invasion that play an essential role in host cell entry. Many microneme proteins (MICs) function in protein complexes, and each complex contains at least one protein that displays a cleavable propeptide. Although MIC propeptides have been implicated in forward targeting to micronemes, the specific amino acids involved have not been identified. It was also not known if the propeptide has a general function in MICs trafficking in T. gondii and other apicomplexans. Here we show that propeptide domains are extensively interchangeable between T. gondii MICs and also with that of Eimeria tenella MIC5 (EtMIC5), suggesting a common mechanism of function. We also performed N-terminal deletion and mutational analysis of M2AP and MIC5 propeptides to show that a valine at position +3 (relative to signal peptidase cleavage) of proM2AP and a leucine at position +1 of proMIC5 are crucial for targeting to micronemes. Valine and leucine are closely related amino acids with similar side chains, implying a similar mode of function, a notion that was confirmed by correct trafficking of TgM2AP-V/L and TgMIC5-L/V substitution mutants. Propeptides of AMA1, MIC3 and EtMIC5 have valine or leucine at or near the N-termini and mutagenesis of these conserved residues validated their role in microneme trafficking. Collectively, our findings suggest that discrete, aliphatic residues at the extreme N-termini of proMICs facilitate trafficking to the micronemes.     

Intramembrane cleavage of microneme proteins at the surface of the apicomplexan parasite Toxoplasma gondii

Apicomplexan parasites actively secrete proteins at their apical pole as part of the host cell invasion process. The adhesive micronemal proteins are involved in the recognition of host cell receptors. Redistribution of these receptor-ligand complexes toward the posterior pole of the parasites is powered by the actomyosin system of the parasite and is presumed to drive parasite gliding motility and host cell penetration. The microneme protein protease termed MPP1 is responsible for the removal of the C-terminal domain of TgMIC2 and for shedding of the protein during invasion. In this study, we used site-specific mutagenesis to determine the amino acids essential for this cleavage to occur. Mapping of the cleavage site on TgMIC6 established that this processing occurs within the membrane-spanning domain, at a site that is conserved throughout all apicomplexan microneme proteins. The fusion of the surface antigen SAG1 with these transmembrane domains excluded any significant role for the ectodomain in the cleavage site recognition and provided evidence that MPP1 is constitutively active at the surface of the parasites, ready to sustain invasion at any time.     

TgM2AP participates in Toxoplasma gondii invasion of host cells and is tightly associated with the adhesive protein TgMIC2

Like other members of the medically important phylum Apicomplexa, Toxoplasma gondii is an obligate intracellular parasite that secretes several classes of proteins involved in the active invasion of target host cells. Proteins in apical secretory organelles known as micronemes have been strongly implicated in parasite attachment to host cells. TgMIC2 is a microneme protein with multiple adhesive domains that bind target cells and is mobilized onto the parasite surface during parasite attachment. Here, we describe a novel parasite protein, TgM2AP, which is physically associated with TgMIC2. TgM2AP complexes with TgMIC2 within 15 min of synthesis and remains associated with TgMIC2 in the micronemes, on the parasite surface during invasion and in the culture medium after release from the parasite plasma membrane. TgM2AP is proteolytically processed initially when its propeptide is removed during transit through the golgi and later while it occupies the parasite surface after discharge from the micronemes. We show that TgM2AP is a member of a protein family expressed by coccidian parasites including Neospora caninum and Eimeria tenella. This phylogenic conservation and association with a key adhesive protein suggest that TgM2AP is a fundamental component of the T. gondii invasion machinery.     

Human erythrocyte band 3 functions as a receptor for the sialic acid-independent invasion of Plasmodium falciparum. Role of the RhopH3–MSP1 complex

Plasmodium falciparum takes advantage of two broadly defined alternate invasion pathways when infecting human erythrocytes: one that depends on and the other that is independent of host sialic acid residues on the erythrocyte surface. Within the sialic acid-dependent (SAD) and sialic acid-independent (SAID) invasion pathways, several alternate host receptors are used by P. falciparum based on its particular invasion phenotype. Earlier, we reported that two putative extracellular regions of human erythrocyte band 3 termed 5C and 6A function as host invasion receptor segments binding parasite proteins MSP1 and MSP9 via a SAID mechanism. In this study, we developed two mono-specific anti-peptide chicken IgY antibodies to demonstrate that the 5C and 6A regions of band 3 are exposed on the surface of human erythrocytes. These antibodies inhibited erythrocyte invasion by the P. falciparum 3D7 and 7G8 strains (SAID invasion phenotype), and the blocking effect was enhanced in sialic acid-depleted erythrocytes. In contrast, the IgY antibodies had only a marginal inhibitory effect on FCR3 and Dd2 strains (SAD invasion phenotype). A direct biochemical interaction between erythrocyte band 3 epitopes and parasite RhopH3, identified by the yeast two-hybrid screen, was established. RhopH3 formed a complex with MSP1₁₉ and the 5ABC region of band 3, and a recombinant segment of RhopH3 inhibited parasite invasion in human erythrocytes. Together, these findings provide evidence that erythrocyte band 3 functions as a major host invasion receptor in the SAID invasion pathway by assembling a multi-protein complex composed of parasite ligands RhopH3 and MSP1.     

A role for coccidian cGMP-dependent protein kinase in motility and invasion

The coccidian parasite cGMP-dependent protein kinase is the primary target of a novel coccidiostat, the trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl] pyridine (compound 1), which effectively controls the proliferation of Eimeria tenella and Toxoplasma gondii parasites in animal models. The efficacy of compound 1 in parasite-specific metabolic assays of infected host cell monolayers is critically dependent on the timing of compound addition. Simultaneous addition of compound with extracellular E. tenella sporozoites or T. gondii tachyzoites inhibited [3H]-uracil uptake in a dose-dependent manner, while minimal efficacy was observed if compound addition was delayed, suggesting a block in host cell invasion. Immunofluorescence assays confirmed that compound 1 blocks the attachment of Eimeria sporozoites or Toxoplasma tachyzoites to host cells and inhibits parasite invasion and gliding motility. Compound 1 also inhibits the secretion of micronemal adhesins (E. tenella MIC1, MIC2 and T. gondii MIC2), an activity closely linked to invasion and motility in apicomplexan parasites. The inhibition of T. gondii MIC2 adhesin secretion by compound 1 was not reversed by treatment with calcium ionophores or by ethanol (a microneme secretagogue), suggesting a block downstream of calcium-dependent events commonly associated with the discharge of the microneme organelle in tachyzoites. Transgenic Toxoplasma strains expressing cGMP-dependent protein kinase mutant alleles that are refractory to compound 1 (including cGMP-dependent protein kinase knock-out lines complemented by such mutants) were used as tools to validate the potential role of cGMP-dependent protein kinase in invasion and motility. In these strains, parasite adhesin secretion, gliding motility, host cell attachment and invasion displayed a reduced sensitivity to compound 1. These data clearly demonstrate that cGMP-dependent protein kinase performs an important role in the host-parasite interaction.     

A thrombospondin structural repeat containing rhoptry protein from Plasmodium falciparum mediates erythrocyte invasion

Host cell invasion by Plasmodium falciparum requires multiple molecular interactions between host receptors and parasite ligands. A family of parasite proteins, which contain the conserved thrombospondin structural repeat motif (TSR), has been implicated in receptor binding during invasion. In this study we have characterized the functional role of a TSR containing blood stage protein referred to as P. falciparum thrombospondin related apical merozoite protein (PfTRAMP). Both native and recombinant PfTRAMP bind untreated as well as neuraminidase, trypsin or chymotrypsin‐treated human erythrocytes. PfTRAMP is localized in the rhoptry bulb and is secreted during invasion. Adhesion of microneme protein EBA175 with its erythrocyte receptor glycophorin A provides the signal that triggers release of PfTRAMP from the rhoptries. Rabbit antibodies raised against PfTRAMP block erythrocyte invasion by P. falciparum suggesting that PfTRAMP plays an important functional role in invasion. Combination of antibodies against PfTRAMP with antibodies against microneme protein EBA175 provides an additive inhibitory effect against invasion. These observations suggest that targeting multiple conserved parasite ligands involved in different steps of invasion may provide an effective strategy forthe development of vaccines against blood stage malaria parasites.     

Structural insights into microneme protein assembly reveal a new mode of EGF domain recognition

The obligate intracellular parasite Toxoplasma gondii, a member of the phylum Apicomplexa that includes Plasmodium spp., is one of the most widespread parasites and the causative agent of toxoplasmosis. Adhesive complexes composed of microneme proteins (MICs) are secreted onto the parasite surface from intracellular stores and fulfil crucial roles in host-cell recognition, attachment and penetration. Here, we report the high-resolution solution structure of a complex between two crucial MICs, TgMIC6 and TgMIC1. Furthermore, we identify two analogous interaction sites within separate epidermal growth factor-like (EGF) domains of TgMIC6-EGF2 and EGF3-and confirm that both interactions are functional for the recognition of host cell receptor in the parasite, using immunofluorescence and invasion assays. The nature of this new mode of recognition of the EGF domain and its abundance in apicomplexan surface proteins suggest a more generalized means of constructing functional assemblies by using EGF domains with highly specific receptor-binding properties.     

Secretion of micronemal proteins is associated with toxoplasma invasion of host cells

Toxoplasma gondii is an obligate intracellular parasite that actively invades a wide variety of vertebrate cells, although the basis of its pervasive cell invasion is not completely understood. Here, we demonstrate, using several independent assays, that Toxoplasma invasion of host cells is tightly coupled to the release of proteins stored within apical secretory granules called micronemes. Both microneme secretion and cell invasion were highly temperature dependent, and partial depletion of microneme resulted in a transient loss of infectivity. Chelation of parasite intracellular calcium strongly inhibited both microneme release and invasion of host cells, and this effect was partially reversed by raising intracellular calcium using the ionophore A23187. We also provide evidence that a staurosporine-sensitive kinase activity regulates microneme discharge and is required for parasite invasion of host cells. Additionally, we demonstrate that, during apical attachment to the host cell, the micronemal protein MIC2 is released at the junction between the parasite and the host cell. During invasion, MIC2 is successively translocated towards the posterior end of the parasite and is shed before entry of the parasite into the vacuole. Furthermore, we show that the full-length cellular form of MIC2, but not the proteolytically modified secreted form of MIC2, binds specifically to host cells. Collectively, these observations strongly imply that micronemal proteins play a role in Toxoplasma invasion of host cells.