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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Subcell Biochem. Author manuscript; available in PMC 2010 March 30.
Published in final edited form as:
Subcell Biochem. 2008; 47: 33–45.
PMCID: PMC2847500
NIHMSID: NIHMS97921

Receptor-ligand interaction and invasion: Microneme proteins in apicomplexans

I. Introduction

The invasive stages (zoites) of most apicomplexan parasites are polarised cells that use their actinomyosin-powered gliding motility or “glideosome” system to move over surfaces, migrate through biological barriers and invade and leave host cells. Central to these processes is the timely engagement and disengagement of specific receptors upon the regulated release of apical invasion proteins from parasite secretory organelles (micronemes, rhoptries). In this short review, we summarise recent progress on identification and functional characterisation of apical invasion proteins mobilised to the parasite surface from the microneme organelles. We have restricted our focus to Toxoplasma, Eimeria, Cryptosporidium and the non-erythrocytic stages of Plasmodium because these organisms have been the most intensively studied apicomplexans that invade nucleated cells and because invasion by erythrocytic stages of Plasmodium is covered in the next chapter.

Micronemes are the smallest of the apicomplexan secretory organelles that cluster at the apical end of the zoite. The number of micronemes varies enormously between different genera, species and developmental stages with those zoites displaying vigorous and extensive gliding or migration activity generally having the most. Thus, Theileria zoites, which are non-motile, do not migrate and do not display active host cell invasion, have no micronemes;1 merozoites of Plasmodium, which neither glide nor migrate but rapidly and actively invade erythrocytes, have few;2 sporozoites and merozoites of Eimeria, which glide, migrate through intestinal contents and actively invade enterocytes have many;3 and Plasmodium ookinetes, which glide and migrate through the midgut epithelium of the mosquito, but do not classically invade host cells, also have many (and by contrast, do not have any rhoptries).4 This long-standing correlation between micronemes and parasite motility, migration and invasion is well supported by a variety of biochemical and genetic studies which show: (1) that microneme secretion is rapidly up-regulated when parasites make contact with host cells;5 (2) that some Plasmodium microneme proteins are targets of erythrocytic invasion-inhibitory antibodies;610 (3) that parasite invasion is blocked when microneme secretion is chemically inhibited;11, 12 and (4) that genes encoding MICs either alone, or in concert with others, are essential for effective parasite motility, migration and invasion.

MICs have been identified in a variety of approaches (reviewed in 1314), most recently through the application of proteomics to gradient-purified organelles and excreted-secreted antigens.15 Figure 1 summarises the current repertoires of MICs, including only those genes for which a full sequence and a verified organellar localisation is known. The majority of MICs comprise multiple copies of a limited number of adhesive domain types, which has allowed the identification of a large number of additional putative microneme proteins bearing these domains in the parasite databases.16, 17 Based on this it is likely that many more proteins will be shown to occupy the micronemes in future studies.

Figure 1
Modular MICs.

II. Ligand domains and their cellular receptors

1. Thrombospondin-1 type 1 domains (TSR)

Thrombospondin-1 (TSP-1) is a multifunctional, glycoprotein adhesion molecule that mediates a broad range of biological interactions via three distinct repeated domains designated types 1, 2 and 3.18 The adhesive TSP-1 type 1 domain, TSR, is a small ~60 residue structure found in the extracellular regions of several protein families involved in immunity, cell adhesion and neuronal development, and shown to have binding activity for a number of cellular and matrix molecules (reviewed in 19). The TSR is an ancient eukaryotic module that is found in many nematode and arthropod proteins as well as those from the Apicomplexa.20 One or more copies of the TSR are present in several apicomplexan MICs including Plasmodium thrombospondin-related adhesive protein (TRAP), circumsporozoite protein (CSP), and circumsporozoite-and-TRAP related protein (CTRP); Eimeria EtMIC1, and EtMIC4; Cryptosporodium TRAP-C1 and sporozoite cysteine-rich protein (SCRP) and Toxoplasma TgMIC2 and TgMIC12. Structures of TSR domains from TSP-1,21 F-spondin22 and Plasmodium TRAP,23 representing two different TSR groups in respect to the organisation of their cysteine residues (Groups I and II), have been determined. Despite the different disulphide bonding patterns, these three TSRs share a highly similar elongated structure consisting of an anti-parallel, three-β-stranded fold that is additionally stabilised by stacked tryptophan and arginine residues (Figure 2). A positively charged groove formed by the arginine stack was proposed to be the site of interaction with ligands and receptors, particularly glycosaminoglycans.21 Several studies have shown that MICs containing TSRs bind host ligands2426 and recently chemical-shift mapping experiments, in which low-molecular weight heparin was titrated into 15N-labelled TRAP-TSR, confirmed a site of interaction in the N-terminal half of the domain on the side of the aligned arginines.23 Based on the structure, MICs containing TSRs would be predicted to extend out from the parasite surface after secretion from the micronemes and be thus ideally positioned to engage host surface receptors for attachment and invasion (see below).

Figure 2
Structural features of micronemal ligands.

Toxoplasma TgMIC1 and Eimeria EtMIC3 possess domains previously described as TSR-like, since they share some key sequence features with classical TSRs, but are now known to adopt an unrelated novel fold termed MAR (microneme adhesive repeat) (S. Matthews and D. Soldati, personal communication). The MAR domain is also able to bind host ligands27, 28 (J. Bumstead & F. Tomley, unpublished); TgMIC1 MAR binds specifically to sialic acid (S. Matthews and D. Soldati, personal communication) whereas the orthologous protein, NcMIC1, from Neospora caninum binds glycosaminoglycans.29

MIC TSRs have functions other than cell binding, for example the TSRs of TgMIC2 are implicated its tight association with its partner protein MIC2 associated protein, TgM2AP (J Harper & V. Carruthers, unpublished) and the TSR-like domains of TgMIC1 recruit and interact with TgMIC4 in the TgMIC1-4-6 complex.28

2. Von Willebrand A domain/integrin inserted (I) domains

The inserted (I) domain is found in the α- and β-chains of several vertebrate cell-surface integrins and is homologous to the von Willebrand A (WVA) domain, which is present in many extracellular matrix proteins. This ~200 residue A/I domain is ancient, found in proteins derived from eukaryotes, eubacteria and archaebacteria,30 and adopts a Rossman dinucleotide binding fold consisting of five parallel and one anti-parallel β-strands that collectively are sandwiched by 7 α-helices (Figure 2). In many A/I domains, a non-continguous motif of amino acids is exposed on the surface of the structure to form a metal ion-dependent adhesion motif (MIDAS).31, 32 Although the MIDAS is crucial for binding in some cases, such as in type VI collagen dimerisation33, it appears irrelevant in others, such as in binding of the third VWA domain of von Willebrand Factor to fibrillar collagen.34 Several TSR-containing apicomplexan MICs possess one or more A/I domain, and the MIDAS sequence is generally well conserved in these. Experimental studies have shown that the function of these apicomplexan A/I domains may be mediated by both MIDAS dependent and independent mechanisms. Thus, mutations in the MIDAS of TRAP of Plasmodium berghei affect parasite invasion activity,35 and binding of this domain to hepatocytes and to fetuin is MIDAS dependent;36 however, binding to glycosaminoglycans is not mediated by MIDAS 37. Similarly, the A/I domain of Toxoplasma TgMIC2 binds heparin in a MIDAS-independent manner.38 Interestingly, exhaustive searching of the databases of Cryptosporidium has failed to identify any proteins containing A/I domains in this member of the phylum (T. Templeton, personal communication).

3. Apple/PAN domains

Apple domains, which are a subset of the plasminogen, apple, nematode (PAN) superfamily, have been identified in plasminogen-related proteins such as coagulation factor XI, plasma pre-kallikrein, hepatocyte growth factor, macrophage stimulation factor and also in several nematode proteins. Apple/PAN domains have three conserved disulphide bridges that are essential for their tertiary structure, but homology in the primary amino acid sequence between domains is generally low, which may contribute to their very different and highly specific ligand binding properties. For example the four Apple/PAN domains from FXI display very different ligand specificities: A1 binds the FXI co-factor H-kininogen and thrombin,39, 40 A2 binds the FXI substrate, FIX,41 A3 also binds FIX and heparin42, 43 and A4 binds FXIIa.44 Apple/PAN domains are present in several apicomplexan MIC proteins including EtMIC5, TgMIC4 and TRAP-CI and the solution structure of a single (A9) domain from Eimeria tenella confirmed its structural homology to the Apple/PAN superfamily45 (Figure 2). Very recently the crystal structures of apical membrane antigens (AMA1) of Plasmodium species revealed that the two most N-terminal domains of these MICs are also highly divergent members of the Apple/PAN superfamily46, 47 (Figure 2). Most functional information on apicomplexan Apple/PAN domains has come from the study of TgMIC4, which contains 6 tandem domains and which exists as a structural heterocomplex with TgMIC1 and TgMIC6.48, 49 The first two Apple domains of TgMIC4 interact directly with the twin TSR-like domains of TgMIC1 and in the absence of TgMIC1 binding of TgMIC4 to host cells is almost entirely ablated;28 however, is not known whether this is due to incorrect folding under these conditions or to the inherent lack of cell binding properties of TgMIC4. Interestingly NcMIC4, an orthologue of TgMIC4 in the closely related parasite Neospora caninum, is able to bind lactose, a property that is not shared by TgMIC4, which does not bind.50 The function and binding properties of the newly defined Apple/PAN domains of AMA1 are not well defined, although recent data from Toxoplasma indicates that TgAMA1 co-operates with rhoptry neck proteins in the formation and maintenance of the moving junction during host cell invasion.51, 52

4. EGF-like domains

EGF-like domains are widely distributed in membrane-bound and extracellular eukaryotic proteins and are involved in many different and diverse biological functions including blood coagulation, cell signalling, cell migration and maintenance of extracellular matrix architecture. These domains typically consist of ~50 amino acids with three conserved disulphide bridges and a subclass of EGF-like domains that bind calcium (cbEGFs) has been identified that have a conserved D/N-x-D/N-E/Q-xm-D/N*-xn-Y/F motif, (where m and n are variable and * indicates β-hydroxylation.53 The first apicomplexan proteins containing EGF-like domains to be identified were GPI-linked proteins from Plasmodium54, 55 but more recently a number of apicomplexan MICs with EGF-like domains have been studied including SCRP, EtMIC4, and TgMICs 3, 6, 7, 8, 9, and 12. So far no EGF-like containing MICs have been found in Plasmodium species. The majority of the domains described in apicomplexan MICs are regular EGF-like, but the cbEGF motif is present in 22 of the 31 EGF-like domains of EtMIC456 and its homologue TgMIC12 (ToxoDB 57.m01872; F. Stavru and D. Soldati, personal communication). Study of cbEGFs in EtMIC4 has shown that in the presence of calcium these domains adopt a proteinase-resistant, extended structure that would favour the interaction of the N-terminal portion of the molecule with host cell ligands.57 Interestingly, EtMIC4 forms a stable very high molecular mass heteromeric complex with the soluble Apple/PAN domain containing protein EtMIC5 although the precise sites of interaction between these two MICs are not yet mapped (J. Periz & F. Tomley, unpublished). In Toxoplasma, TgMIC3 contains both EGF-like and lectin-like domains and binds to all nucleated cells tested as well as to the tachyzoite surface.58 The receptor-binding properties of TgMIC3 are attributed to the lectin-like domain, whereas the EGF-like domains are proposed to promote proper folding of the protein in order to expose the binding regions. In addition, they may be involved in heteromeric polymerisation with the transmembrane MIC TgMIC8, which contains 10 EGF-like domains and which functions as an ‘escorter’ to ensure delivery of TgMIC3 to the micronemes. Similarly, in the TgMIC1-4-6 adhesive complex it is the transmembrane, EGF-like domain containing TgMIC6 that is responsible for targeting to the micronemes but in this case oligomerisation is promoted and stabilised by the interaction of the third EGF of TgMIC6 domain, together with its downstream acidic region, with the galectin domain of TgMIC1.28

5. Lectin domains

Two types of domains related to lectins have been identified within apicomplexan MICs. Chitin-binding like (CBL) domains are found in a variety of plant lectins including plant defensins that have anti-fungal chitinase activitiy. CBLs are typically composed of ~ 30–43 amino acids with four conserved disulphide bridges and several conserved aromatic residues that mediate binding of the domain to N-acetyl glucosamine.59 CBLs with lectin (or agglutinin) properties are able to bind and cross link GlcNAc-containing polymers and in Toxoplasma, TgMIC3 and TgMIC8 each contain a single CBL-domain at their N-termini, followed by several EGF-like domains.58, 60 Binding of the CBL-domain of TgMIC3 to host cell surfaces is dependent upon its dimerisation, which is mediated by the interaction of the C-terminal regions of each monomer61, and disruption of the CBL aromatic residues presumed to be important for binding results in lowered parasite virulence.62 Fusion of the TgMIC3 dimerisation domain to the extracellular domain of TgMIC8 promotes dimerisation and binding of the chimera, indicating that the CBL of TgMIC8 also possesses binding activity when in a dimeric form.62

Another lectin-related domain in an apicomplexan MIC was recently identified from the three-dimensional structure of the C-terminal domain of Toxoplasma TgMIC1.28 This domain has a galectin-like fold, which consists of a β-barrel formed by the association of two multi-stranded β-sheets. Galectins are soluble, calcium-independent, carbohydrate-binding animal lectins, however the critical side chains that mediate lectin activity are not conserved in the TgMIC1 galectin domain and no detectable binding to a range of carbohydrate substrates was observed in NMR chemical shift mapping experiments. Instead, the TgMIC1 galectin domain displays a large hydrophobic surface reminiscent of the protein-protein interaction domains seen in bacterial class I chaperones of the type three secretion system and both NMR and biochemical studies indicate that during the biogenesis of the TgMIC1-4-6 adhesive complex, this domain recruits and stabilises TgMIC6 providing a highly specific quality control mechanism for the exit of TgMIC6 from the ER/Golgi and for subsequent trafficking of the adhesive complex to the micronemes.

III. Adhesive Complexes: Assembly and Organization

1. Propensity to form oligomers

Adhesive proteins often form oligomeric complexes with themselves or other proteins that contribute to adhesion or serve a regulatory function. For example, cadherins are a family of vertebrate adhesive proteins expressed as homodimers that strengthen cell-cell junctions. Integrins are heterodimeric, transmembrane glycoproteins primarily responsible for mediating cell interactions with extracellular matrix (ECM). The propensity to form oligomeric adhesive complexes has been demonstrated in several apicomplexans, although most of the mechanistic studies have been done in Toxoplasma. Oligomerization bestows adhesive proteins with several important advantages.

First, oligomerization can promote the proper folding of proteins in a complex, as recently shown for the TgMIC1-4-6 complex.28 TgMIC1 is a soluble protein that simultaneously associates with TgMIC4 through its two TSR-like MAR domains and with the transmembrane escorter protein TgMIC6 through its C-terminal (CT) galectin-like domain. As mentioned above, NMR spectroscopy revealed that the CT domain is incapable of binding sugars but instead forms an interface with the third EGF-like domain of TgMIC6, which also contains an acidic element (TgMIC6-EGF3acid).28 When mixed together and monitored by NMR, the TgMIC1 CT domain facilitated the folding and stabilization of the TgMIC6-EGF3acid. The TgMIC1 CT domain also rescued the secretory retention phenotype of TgMIC6 in mic1KO parasites, presumably by navigating through the quality control system that recognizes misfolded proteins. These findings reveal new molecular insights into the interdependence of adhesive proteins for correct folding and movement through the secretory pathway.

Second, assembly into protein complexes allows cooperation in trafficking to the micronemes. MIC complexes typically have one transmembrane (TM) protein. These TM MICs are also referred to as escorters since they accompany and guide the other soluble members to the micronemes based on the targeting signals in their C-terminal tails.49, 60 In TgMIC2, this signal is provided by two tyrosine-based sorting motifs capable of directing a heterologous protein to the micronemes.63 Genetic disruption of any of the TM MICs results in retention of the other members of the complex along the secretory system or in mistargeting to the default secretory pathway, which in T. gondii is secretion via the dense granules. When the level of TgMIC2 expression is experimentally reduced, TgM2AP colocalizes with the dense granules and is secreted into the PV.64 Similarly, TgMIC6 knockout parasites show a complete misrouting of TgMIC1 and TgMIC4 to the dense granules.49 Nonetheless, escorters still depend on their cargo for proper trafficking since soluble proteins in the complexes are required for protein folding, as is the function of the galectin-like domain of TgMIC1,28 or necessary for exiting an endosomal compartment associated with microneme biogenesis, as shown for the TgM2AP propeptide (Harper et al., submitted).

Third, different combinations of partners can expand the receptor repertoire and/or fine-tune the specificity of receptor binding. Humans express eighteen integrin [alpha]-subunits and eight β-subunits that form 24 heterodimers for recognition of distinct but overlapping receptors.65, 66 Although there are no firm examples of subunit mixing in the apicomplexa, these parasites often express paralogous families of adhesive proteins with the potential to participate in such a phenomenon. Four closely related putative adhesins were recently identified in a proteomic screen of Toxoplasma secretory proteins.67 These proteins have four Apple/PAN domains but no predicted anchoring sequence, and, by analogy with TgMIC4 and its association with TgMIC1 and TgMIC6, they likely oligomerize with a TM protein, possibly in a manner that would expand their receptor binding capabilities. Three additional genes coding for proteins closely related to TgMIC1 are also present in the Toxoplasma genome (D. Soldati, personal communication).

Fourth, oligomerization allows proteins from distinct compartments to facilitate invasion collaboratively. Two studies51, 52 have recently shown that the microneme protein TgAMA1 oligomerizes with three proteins derived from the rhoptry neck: TgRON2, TgRON4, and TgRON5. Although they are discharged from different organelles during invasion, TgAMA1 and TgRON2/4/5 form an oligomeric complex on the parasite surface within the moving junction, a ring-like constriction that slides over the parasite as it penetrates the host cell. TgAMA1 is a key component of the complex since depletion of this protein causes a failure to form the moving junction and parasite invasion is arrested at the stage of apical attachment.68 Since RON4 is predicted to be an integral membrane protein, this raises the hypothesis that it inserts into the host plasma membrane and acts as an autologous receptor for cell invasion.51 In this case, oligomerization would allow the parasite to use its own receptor to support invasion of the many cell types susceptible to Toxoplasma invasion.

Finally, oligomerization increases valency and avidity, thereby enhancing the formation of a robust binding interface. For example, TgMIC2-M2AP is a heterohexameric complex consisting of a trimer of dimers.38, 69 The corresponding complex in Eimeria tenella, EtMIC1-MIC2, presumably also forms a similar hexameric assembly. Such an arrangement could promote tight binding to a complementary oligomeric receptor on the host cell surface, thereby allowing the parasite to grip sufficiently well to power its way into the target cell.

2. Ligand organization in micronemes and on parasite surface

It is not known precisely how adhesive ligands are organized within micronemes. However, several features suggest that ligands are packaged in an orderly fashion. First, the contents of Cryptosporidium micronemes are arranged in an array of 15 nm cubic crystals arranged in a pine-cone-like pattern.70 Although this crystalline appearance is unique to Cryptosporidium, micronemes of other apicomplexa are electron dense, implying a high protein concentration. Second, since a number of micronemes are discharged in rapid succession, a strong measure of organization is presumably necessary to achieve efficient deployment. Finally, the internal dimensions of micronemes (~75nm × 150nm) cannot accommodate some of the larger microneme proteins (e.g., EtMIC4-MIC5) in their fully extended state (see also below) and therefore these proteins must be packaged in orderly fashion so that they are primed for secretion onto the parasite surface.

It has been proposed that some microneme proteins are involved in organizing the organellar contents. For example, TgMIC10 and TgMIC11 are small, soluble microneme proteins that display a marked charge asymmetry, which may promote electrostatic assembly into higher ordered structures.71, 72 Unlike most other microneme proteins, TgMIC10 and TgMIC11 do not associate with the parasite surface during invasion, consistent with an alternative role independent of adhesion. During transport to the micronemes, TgMIC11 is proteolytically processed to remove an internal propeptide in a manner reminiscent of insulin maturation within nascent secretory granules of pancreatic beta cells. Insulin processing is thought to promote its ordered packaging and retention in maturing secretory granules,73, 74 although this idea is somewhat controversial.75

During gliding and invasion the microneme contents are deployed onto the parasite’s apical surface where substrate or receptor engagement occurs. Adhesive complexes are not randomly distributed. For example, TRAP is arranged in a cap or ring-like pattern on gliding Plasmodium sporozoites 76. Also, the EtMIC4-MIC5 complex displays a punctate pattern on the surface of invading Eimeria sporozoites77 in a manner similar to TgMIC2-M2AP during Toxoplasma tachyzoite invasion 78. Invading zoites display a particularly high density of ligands at the external boundary of the moving junction. The organization of ligands in this adhesion zone may further promote multivalent, high avidity interactions with host receptors, especially if the receptors have a complementary clustering distribution. Clustering may therefore be an additional level of organization that further promotes the creation of a robust binding interface between the parasite and host cell membranes.

IV. The surface ligand landscape: does size (and conformation) matter?

Crystal structure analysis of several domain types found in apicomplexan microneme proteins is beginning to reveal both the approximate size and shape of these important ligands. For example, cbEGF domains form an elongated structure that is stabilized by interdomain Ca2+ binding and hydrophobic interactions between adjacent domains.53 Since the majority of EGF domains in the extracellular portion of EtMIC4 are of the cbEGF type, EtMIC4 is predicted to adopt a highly extended conformation that could project nearly 400nm from the parasite surface. However, it is unlikely that this structure is completely rigid there is greater flexibility between non-calcium binding EGF domains.57 This semi-rigid conformation may allow the molecule to project maximally from the membrane while still retaining some degree of flexibility to “survey” the host cell surface for receptors. The ninth Apple/PAN domain of EtMIC5 adopts a globular α/β structure with the N- and C-termini situated on the same side of the molecule.45 Although for EtMIC5 the structure of only one domain was solved, Plasmodium AMA1 has two PAN/Apple domains that are stacked upon one another,47 suggesting that EtMIC5 and other multi-PAN/Apple domain containing microneme proteins may also adopt an elongated structure that projects away from the parasite surface. Based on the crystal structures of the A/I domains from various integrins and a pair of TSR domains from thrombospondin, TRAP family members including TgMIC2 are predicted to form a “ball-on-a-stick” type of structure that could extend ~80nm from the parasite surface. Six tandem TSR domains that form a highly elongated stalk provide most of the molecule’s height. The trimeric arrangement likely imparts a high degree of rigidity and strength in the molecule, which may be important to form a solid connection between extracellular receptors and the parasite’s intracellular motility system.

For mammalian cell adhesion, recent studies have also provided new insight into role of conformational shifting in modulating ligand affinity. Molecular electron microscopy of the integrin α5β1 showed that it undergoes a dramatic conformational shift from a “closed” to “open” configuration upon activation by inside-out signaling and/or exposure to certain divalent cations.79, 80 As shown in Figure 2, in the closed, low affinity position the heterodimer is bent over with the paired A/I domans positioned proximal to the cell membrane. However, when Mn2+ binds to the MIDAS site the complex “stands up” to project the adhesive A/I domains 2–3 times further away from the cell membrane. Although no direct evidence is available, similarly dramatic conformational changes could occur in micronemal ligands. For example, if EtMIC4 is not exposed to high concentrations of Ca2+ during transport and packaging in the micronemes then it would be sufficiently flexible and compact to fit within the microneme lumen. However, upon secretion and exposure to millimolar concentrations of Ca2+ in the extracellular milieu, EtMIC4 might unfurl to attain maximum height for long-range interactions with host receptors in the initial apical docking of the parasite. Other large microneme proteins in Cryptosporidium (CpGP900) and Toxoplasma (TgMIC12) may play a similar role. In this manner the parasite could establish an initial connection between its apical pole and the host surface before using other perhaps higher affinity or more abundant micronemal ligands to strengthen the grip for active penetration.

V. Role of micronemal proteins in migration across biological barriers

For Plasmodium, the mosquito midgut and the sinusoidal layer of the liver are two significant biological barriers against infection and cell migration activity is needed for the zoites to breach these barriers.

Ookinetes of Plasmodium are highly motile and they migrate through the midgut epithelium of the mosquito causing massive destruction. The microneme proteins CTRP, SOAP (soluble ookinete adhesive protein), MAOP (membrane attack ookinete protein) and CelTOS (cell-traversal protein for ookinetes and sporozoites) have been shown to play crucial roles. CTRP is essential for apical attachment to the midgut epithelial cell,81 SOAP is involved in mosquito midgut invasion and oocyst development,82 MAOP which has a MACPF domain is necessary for ookinetes to breach the apical plasma membrane of the epithelial cell83 and CelTOS is needed for the ookinetes to migrate through the cell cytoplasm to reach the basal lamina where oocyst development occurs.84

Sporozoites of Plasmodium are able to glide, migrate and invade host cells. Entry of the sporozoite into the hepatocyte is controversial and has been reported to occur following direct parasite migration through cells and by ‘classical’ invasion, vacuole formation and egress. It has been suggested that sporozoite migration through hepatocytes has an effect on subsequent sporozoite infectivity for new hepatocytes and on permissiveness of surrounding hepatocytes (via release of hepatocyte growth factor, HGF).85, 86 However, gene-targeting experiments on sporozoite microneme proteins contradict this - SPECT (Sporozoite-protein-essential-for-cell-traversal) disrupted sporozoites are deficient in cell migration yet they show normal cell invasion and gliding motility.87 This indicates that cell migration is not an absolute requirement for cell invasion, although it is clearly important in vivo since disruption of SPECT decreases liver infectivity ~20-fold. This decrease in infectivity was reversed by depletion of Kupfer cells that line the liver sinusoids, leading to the conclusion that the cell migration activity mediated by SPECT is required to cross the liver sinusoidal barrier.87 Two other sporozoite MICs are implicated in liver invasion. SPECT2 contains a membrane attack complex/perforin domain and disruptants show the same phenotype as SPECT disruptants, thus SPECT2 is presumed also to be necessary for sporozoite traversal of the liver sinusoid.88 CelTOS is expressed in both ookinetes and sporozoites and again disruption of the gene gives essentially the same phenotype as SPECT and SPECT2 except that the disruptants maintain a low level of cell migration (cell wounding) activity.84 It is unclear whether the proteins involved in cell migration function by binding specific receptors on the host cell surface or within the cytoplasm, or whether they function in a regulatory or sensory role (for more details see chapter IX, Frevert et. al.).

While Toxoplasma tachyzoites have not been reported to migrate through cells, recent studies suggest that they cross biological barriers by a paracellular route i.e., between host cells, using ICAM1 as a receptor.89 TgMIC2 was shown to bind ICAM, but only upon proteolytic removal of a short N-terminal extension that preceeds the A/I domain. This proteolytic trimming phenomenon, mediated by a hypothetical surface protease called MPP2, constitutes another means of regulating adhesive activity associated with parasite migration and possibly also attachment.

VI. Summary

Microneme secretion supports several key cellular processes including gliding motility, active cell invasion and migration through cells, biological barriers, and tissues. The modular design of microneme proteins enables these molecules to assist each other in folding and passage through the quality control system, accurately target to the micronemes, oligimerizing with other parasite proteins, and engaging a variety of host receptors for migration and cell invasion. Structural and biochemical analyses of MIC domains is providing new perspectives on how adhesion is regulated and the potentially distinct roles MICs might play in long or short range interactions during parasite attachment and entry. New access to complete genome sequences and ongoing advances in genetic manipulation should provide fertile ground for refining current models and defining exciting new roles for MICs in apicomplexan biology.

Abbreviations

AMA1
apical membrane antigen-1
cbEGF
calcium binding EGF
CelTOS
cell-traversal protein for ookinetes and sporozoites
CBL
chitin-binding-like
CSP
circumsporozoite protein
CTRP
circumsporozoite-and-TRAP related protein
CT
C-terminal
EGF
epidermal growth factor
MAOP
membrane attack ookinete protein
MIDAS
metal ion-dependent adhesion motif
M2AP
MIC2-associated protein
MAR
microneme adhesive repeat
MICs
microneme proteins
PAN
plasminogen apple, nematode
SOAP
soluble ookinete adhesive protein
SCRP
sporozoite cysteine-rich protein
SPECT
sporozoite-protein-essential-for-cell-traversal
TSP-1
thrombospondin-1
TSR
thrombospondin-1 type 1 domain
TRAP
thrombospondin-related adhesive protein
WVA
Willebrand A

VII. References

1. Shaw MK. The same but different: the biology of Theileria sporozoite entry into bovine cells. Int J Parasitol. 1997;27(5):457–474. [PubMed]
2. Langreth SG, Jensen JB, Reese RT, Trager W. Fine structure of human malaria in vitro. J Protozool. 1978;25(4):443–452. [PubMed]
3. Scholtyseck E, Mehlhorn H. Ultrastructural study of characteristic organelles (paired organelles, micronemes, micropores) of sporozoa and related organisms. Z Parasitenkd. 1970;34(2):97–127. [PubMed]
4. Sinden RE, Hartley RH, Winger L. The development of Plasmodium ookinetes in vitro: an ultrastructural study including a description of meiotic division. Parasitology. 1985;91(Pt 2):227–244. [PubMed]
5. Carruthers VB, Sibley LD. Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts. Eur J Cell Biol. 1997;73(2):114–123. [PubMed]
6. Healer J, Murphy V, Hodder AN, et al. Allelic polymorphisms in apical membrane antigen-1 are responsible for evasion of antibody-mediated inhibition in Plasmodium falciparum. Molecular Microbiology. 2004;52(1):159–168. [PubMed]
7. Miller LH, Hudson D, Haynes JD. Identification of Plasmodium knowlesi erythrocyte binding proteins. Mol Biochem Parasitol. 1988;31(3):217–222. [PubMed]
8. Narum DL, Haynes JD, Fuhrmann S, et al. Antibodies against the Plasmodium falciparum Receptor Binding Domain of EBA-175 Block Invasion Pathways that do not Involve Sialic Acids. Infect Immun. 2000;68(4):1964–1966. [PMC free article] [PubMed]
9. Sim BKL, Orlandi PA, Haynes JH, et al. Primary structure of the 175K Plasmodium falciparum erythrocyte binding antigen and identification of a peptide which elicits antibodies that inhibit malaria merozoite invasion. J Cell Biol. 1990;111:1877–1884. [PMC free article] [PubMed]
10. Singh AP, Puri SK, Chitnis CE. Antibodies raised against receptor-binding domain of Plasmodium knowlesi Duffy binding protein inhibit erythrocyte invasion. Mol Biochem Parasitol. 2002;121(1):21–31. [PubMed]
11. Carruthers VB, Giddings OK, Sibley LD. Secretion of micronemal proteins is associated with Toxoplasma invasion of host cells. Cell Microbiol. 1999;1(3):225–235. [PubMed]
12. Wiersma HI, Galuska SE, Tomley FM, Sibley LD, Liberator PA, Donald RGK. A role for coccidian cGMP-dependent protein kinase in motility and invasion. International Journal for Parasitology. 2004;34(3):369–380. [PubMed]
13. Soldati D, Dubremetz JF, Lebrun M. Microneme proteins: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii. Int J Parasitol. 2001;31(12):1293–1302. [PubMed]
14. Tomley FM, Soldati DS. Mix and match modules: structure and function of microneme proteins in apicomplexan parasites. Trends Parasitol. 2001;17(2):81–88. [PubMed]
15. Bromley E, Leeds N, Clark J, et al. Defining the protein repertoire of microneme organelles in the apicomplexan parasite secretor or Eimeria tenella. Proteomics. 2003;3(8):1553–1561. [PubMed]
16. Deng M, Templeton TJ, London NR, Bauer C, Schroeder AA, Abrahamsen MS. Cryptosporidium parvum genes containing thrombospondin type 1 domains. Infect Immun. 2002;70(12):6987–6995. [PMC free article] [PubMed]
17. Templeton TJ, Lancto CA, Vigdorovich V, et al. The Cryptosporidium oocyst wall protein is a member of a multigene family and has a homolog in Toxoplasma. Infect Immun. 2004;72(2):980–987. [PMC free article] [PubMed]
18. Lawler J, Hynes RO. The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins. J Cell Biol. 1986;103(5):1635–1648. [PMC free article] [PubMed]
19. Adams JC, Tucker RP. The thrombospondin type 1 repeat (TSR) superfamily: Diverse proteins with related roles in neuronal development. Developmental Dynamics. 2000;218(2):280–299. [PubMed]
20. Tucker RP. The thrombospondin type 1 repeat superfamily. International Journal of Biochemistry & Cell Biology. 2004;36(6):969–974. [PubMed]
21. Tan KM, Duquette M, Liu JH, et al. Crystal structure of the TSP-1 type 1 repeats: a novel layered fold and its biological implication. Journal of Cell Biology. 2002;159(2):373–382. [PMC free article] [PubMed]
22. Paakkonen K, Tossavainen H, Permi P, et al. Solution structures of the first and fourth TSR domains of F-spondin. Proteins. 2006;64(3):665–72. [PubMed]
23. Tossavainen H, Pihajamaa T, Huttunen TK, et al. The layered fold of the TSR domain of P. falciparum TRAP contains a heparin binding site. Protein Science. 2006;15:1760–1768. [PubMed]
24. Robson KJ, Frevert U, Reckmann I, et al. Thrombospondin-related adhesive protein (TRAP) of Plasmodium falciparum: expression during sporozoite ontogeny and binding to human hepatocytes. Embo J. 1995;14(16):3883–3894. [PubMed]
25. Spaccapelo R, Naitza S, Robson KJ, Crisanti A. Thrombospondin-related adhesive protein (TRAP) of Plasmodium berghei and parasite motility. Lancet. 1997;350(9074):335. [PubMed]
26. Sultan AA, Thathy V, Frevert U, et al. TRAP is necessary for gliding motility and infectivity of Plasmodium sporozoites. Cell. 1997;90(3):511–522. [PubMed]
27. Fourmaux MN, Achbarou A, MercereauPuijalon O, et al. The MIC1 microneme protein of Toxoplasma gondii contains a duplicated receptor-like domain and binds to host cell surface. Molecular and Biochemical Parasitology. 1996;83(2):201–210. [PubMed]
28. Saouros S, Edwards-Jones B, Reiss M, et al. A novel galectin-like domain from Toxoplasma gondii micronemal protein 1 assists the folding, assembly, and transport of a cell adhesion complex. Journal of Biological Chemistry. 2005;280(46):38583–38591. [PubMed]
29. Keller N, Naguleswaran A, Cannas A, et al. Identification of a Neospora caninum microneme protein (NcMIC1) which interacts with sulfated host cell surface glycosaminoglycans. Infect Immun. 2002;70(6):3187–3198. [PMC free article] [PubMed]
30. Tuckwell D. Evolution of von Willebrand factor A (VWA) domains. Biochemical Society Transactions. 1999;27:835–840. [PubMed]
31. Humphries M, Liddington R. Molecular basis of integrin-dependent cell adhesion. In: Kleanthous C, editor. Protein-protein recognition. Oxford UK: Oxford University Press; 2000.
32. Whittaker CA, Hynes RO. Distribution and evolution of von Willebrand/integrin a domains: Widely dispersed adhesion and elsewhere. Molecular Biology of the Cell. 2002;13(10):3369–3387. [PMC free article] [PubMed]
33. Ball S, Bella J, Kielty C, Shuttleworth A. Structural basis of type VI collagen dimer formation. Journal of Biological Chemistry. 2003;278(17):15326–15332. [PubMed]
34. Romijn RAP, Bouma B, Wuyster W, et al. Identification of the collagen-binding site of the von Willebrand factor A3-domain. Journal of Biological Chemistry. 2001;276(13):9985–9991. [PubMed]
35. Matuschewski K, Nunes AC, Nussenzweig V, Menard R. Plasmodium sporozoite invasion into insect and mammalian cells is directed by the same dual binding system. Embo J. 2002;21(7):1597–1606. [PubMed]
36. Jethwaney D, Lepore T, Hassan S, et al. Fetuin-A, a hepatocyte-specific protein that binds Plasmodium berghei thrombospondin-related adhesive protein: a potential role in infectivity. Infection and Immunity. 2005;73(9):5883–5891. [PMC free article] [PubMed]
37. McCormick CJ, Tuckwell DS, Crisanti A, Humphries MJ, Hollingdale MR. Identification of heparin as a ligand for the A-domain of Plasmodium falciparum thrombospondin-related adhesion protein. Mol Biochem Parasitol. 1999;100(1):111–124. [PubMed]
38. Harper JM, Hoff EF, Carruthers VB. Multimerization of the Toxoplasma gondii MIC2 integrin-like A-domain is required for binding to heparin and human cells. Mol Biochem Parasitol Apr. 2004;134(2):201–212. [PubMed]
39. Baglia FA, Jameson BA, Walsh PN. Localization of the high molecular weight kininogen binding site in the heavy chain of human factor XI to amino acids phenylalanine 56 through serine 86. J Biol Chem. 1990;265(7):4149–4154. [PubMed]
40. Baglia FA, Walsh PN. A binding site for thrombin in the apple 1 domain of factor XI. J Biol Chem. 1996;271(7):3652–3658. [PubMed]
41. Baglia FA, Jameson BA, Walsh PN. Identification and chemical synthesis of a substrate-binding site for factor IX on coagulation factor XIa. J Biol Chem. 1991;266(35):24190–24197. [PubMed]
42. Ho DH, Badellino K, Baglia FA, et al. The role of high molecular weight kininogen and prothrombin as cofactors in the binding of factor XI A3 domain to the platelet surface. J Biol Chem. 2000 [PubMed]
43. Sun Y, Gailani D. Identification of a factor IX binding site on the third apple domain of activated factor XI. J Biol Chem. 1996;271(46):29023–29028. [PubMed]
44. Baglia FA, Jameson BA, Walsh PN. Identification and characterization of a binding site for factor XIIa in the Apple 4 domain of coagulation factor XI. J Biol Chem. 1993;268(6):3838–3844. [PubMed]
45. Brown PJ, Mulvey D, Potts JR, Tomley FM, Campbell ID. Solution structure of a PAN module from the apicomplexan parasite Eimeria tenella. J Struct Funct Genomics. 2003;4(4):227–234. [PubMed]
46. Bai T, Becker M, Gupta A, et al. Structure of AMA1 from Plasmodium falciparum reveals a clustering of polymorphisms that surround a conserved hydrophobic pocket. Proceedings of the National Academy of Sciences of the United States of America. 2005;102(36):12736–12741. [PubMed]
47. Pizarro JC, Vulliez-Le Normand B, Chesne-Seck ML, et al. Crystal structure of the malaria vaccine candidate apical membrane antigen 1. Science. 2005;308(5720):408–411. [PubMed]
48. Brecht S, Carruthers VB, Ferguson DJP, et al. The Toxoplasma micronemal protein MIC4 is an adhesin composed of six conserved apple domains. Journal of Biological Chemistry. 2001;276(6):4119–4127. [PubMed]
49. Reiss M, Viebig N, Brecht S, et al. Identification and characterization of an escorter for two secretory adhesins in Toxoplasma gondii. J Cell Biol. 2001;152(3):563–578. [PMC free article] [PubMed]
50. Keller N, Riesen M, Naguleswaran A, et al. Identification and characterization of a Neospora caninum microneme-associated protein (NcMIC4) that exhibits unique lactose-binding properties. Infect Immun. 2004;72(8):4791–4800. [PMC free article] [PubMed]
51. Alexander PL, Mital J, Ward GE, Bradley P, Boothroyd JC. Identification of the moving junction complex of Toxoplasma gondii: a collaboration between distinct secretory organelles. PloS Pathogens. 2005:1e17. [PMC free article] [PubMed]
52. Lebrun M, Michelin A, El Hajj H, et al. The rhoptry neck protein RON4 relocalizes at the moving junction during Toxoplasma gondii invasion. Cellular Microbiology. 2005;7(12):1823–1833. [PubMed]
53. Downing AK, Knott V, Werner JM, Cardy CM, Campbell ID, Handford PA. Solution structure of a pair of calcium-binding epidermal growth factor- like domains: implications for the Marfan syndrome and other genetic disorders. Cell. 1996;85(4):597–605. [PubMed]
54. Blackman MJ, Whittle H, Holder AA. Processing of the Plasmodium falciparum major merozoite surface protein-1: identification of a 33-kilodalton secondary processing product which is shed prior to erythrocyte invasion. Mol Biochem Parasitol. 1991;49(1):35–44. [PubMed]
55. Kaslow DC, Quakyi IA, Syin C, et al. A vaccine candidate from the sexual stage of human malaria that contains EGF-like domains. Nature. 1988;333(6168):74–76. [PubMed]
56. Tomley FM, Billington KJ, Bumstead JM, Clark JD, Monaghan P. EtMIC4: a microneme protein from Eimeria tenella that contains tandem arrays of epidermal growth factor-like repeats and thrombospondin type-I repeats. Int J Parasitol. 2001;31(12):1303–1310. [PubMed]
57. Periz J, Gill AC, Knott V, Handford PA, Tomley FM. Calcium binding activity of the epidermal growth factor-like domains of the apicomplexan microneme protein EtMIC4. Molecular and Biochemical Parasitology. 2005;143(2):192–199. [PubMed]
58. Garcia-Reguet N, Lebrun M, Fourmaux MN, et al. 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. Cell Microbiol. 2000;2(4):353–364. [PubMed]
59. Wright HT, Sandrasegaram G, Wright CS. Evolution of a Family of N-Acetylglucosamine Binding-Proteins Containing the Disulfide-Rich Domain of Wheat-Germ-Agglutinin. Journal of Molecular Evolution. 1991;33(3):283–294. [PubMed]
60. Meissner M, Reiss M, Viebig N, et al. A family of transmembrane microneme proteins of Toxoplasma gondii contain EGF-like domains and function as escorters. J Cell Sci. 2002;115(Pt 3):563–574. [PubMed]
61. Cerede O, Dubremetz JF, Bout D, Lebrun M. The Toxoplasma gondii protein MIC3 requires pro-peptide cleavage and dimerization to function as adhesin. Embo J. 2002;21(11):2526–2536. [PubMed]
62. Cerede O, Dubremetz JF, Soete M, et al. Synergistic role of micronemal proteins in Toxoplasma gondii virulence. Journal of Experimental Medicine. 2005;201(3):453–463. [PMC free article] [PubMed]
63. Di Cristina M, Spaccapelo R, Soldati D, Bistoni F, Crisanti A. Two conserved amino acid motifs mediate protein targeting to the micronemes of the apicomplexan parasite Toxoplasma gondii. Mol Cell Biol. 2000;20(19):7332–7341. [PMC free article] [PubMed]
64. Huynh M-H, Carruthers VB. Toxoplasma MIC2 is a major determinant of invasion and virulence. PLoS Pathogens. 2006;2(8) [epub ahead of print] [PMC free article] [PubMed]
65. Arnaout MA, Goodman SL, Xiong JP. Coming to grips with integrin binding to ligands. Curr Opin Cell Biol. 2002;14(5):641–651. [PubMed]
66. Sonnenberg A. Integrins and their ligands. Curr Top Microbiol Immunol. 1993;184:7–35. [PubMed]
67. Zhou XW, Kafsack BFC, Cole RN, Beckett P, Shen RF, Carruthers VB. The opportunistic pathogen Toxoplasma gondii deploys a diverse legion of invasion and survival proteins. Journal of Biological Chemistry. 2005;280(40):34233–34244. [PMC free article] [PubMed]
68. Mital J, Meissner M, Soldati D, Ward GE. Conditional expression of Toxoplasma gondii apical membrane antigen-1 (TgAMA1) demonstrates that TgAMA1 plays a critical role in host cell invasion. Mol Biol Cell. 2005;16(9):4341–4349. [PMC free article] [PubMed]
69. Jewett TJ, Sibley LD. The Toxoplasma proteins MIC2 and M2AP form a hexameric complex necessary for intracellular survival. J Biol Chem. 2004;279(10):9362–9369. [PubMed]
70. Petry F, Harris JR. Ultrastructure, fractionation and biochemical analysis of Cryptosporidium parvum sporozoites. Int J Parasitol. 1999;29(8):1249–1260. [PubMed]
71. Harper JM, Zhou XW, Pszenny V, Kafsack BF, Carruthers VB. The novel coccidian micronemal protein MIC11 undergoes proteolytic maturation by sequential cleavage to remove an internal propeptide. Int J Parasitol. 2004;34(9):1047–1058. [PubMed]
72. Hoff EF, Cook SH, Sherman GD, et al. Toxoplasma gondii: molecular cloning and characterization of a novel 18-kDa secretory antigen, TgMIC10. Exp Parasitol. 2001;97(2):77–88. [PubMed]
73. Kuliawat R, Prabakaran D, Arvan P. Proinsulin endoproteolysis confers enhanced targeting of processed insulin to the regulated secretory pathway. Mol Biol Cell. 2000;11(6):1959–1972. [PMC free article] [PubMed]
74. Zhang B, Chang A, Kjeldsen TB, Arvan P. Intracellular retention of newly synthesized insulin in yeast is caused by endoproteolytic processing in the Golgi complex. J Cell Biol. 2001;153(6):1187–1198. [PMC free article] [PubMed]
75. Arvan P, Halban PA. Sorting ourselves out: seeking consensus on trafficking in the beta-cell. Traffic. 2004;5(1):53–61. [PubMed]
76. Kappe S, Bruderer T, Gantt S, Fujioka H, Nussenzweig V, Menard R. Conservation of a gliding motility and cell invasion machinery in Apicomplexan parasites. J Cell Biol. 1999;147(5):937–944. [PMC free article] [PubMed]
77. Brown PJ, Billington KJ, Bumstead JM, Clark JD, Tomley FM. A microneme protein from Eimeria tenella with homology to the Apple domains of coagulation factor XI and plasma pre-kallikrein. Mol Biochem Parasitol. 2000;15107(1):91–102. [PubMed]
78. Carruthers VB, Blackman MJ. A new release on life: emerging concepts in proteolysis and parasite invasion. Mol Microbiol. 2005;55(6):1617–1630. [PubMed]
79. Luo BH, Springer TA. Integrin structures and conformational signaling. Curr Opin Cell Biol. 2006 [ebup ahead of print] [PMC free article] [PubMed]
80. Springer TA, Wang JH. The three-dimensional structure of integrins and their ligands, and conformational regulation of cell adhesion. Adv Protein Chem. 2004;68:29–63. [PubMed]
81. Dessens JT, Beetsma AL, Dimopoulos G, et al. CTRP is essential for mosquito infection by malaria ookinetes. Embo J. 1999;18(22):6221–6227. [PubMed]
82. Dessens JT, Siden-Kiamos I, Mendoza J, et al. SOAP, a novel malaria ookinete protein involved in mosquito midgut invasion and oocyst development. Mol Microbiol. 2003;49(2):319–329. [PubMed]
83. Kadota K, Ishino T, Matsuyama T, Chinzei Y, Yuda M. Essential role of membrane-attack protein in malarial transmission to mosquito host. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(46):16310–16315. [PubMed]
84. Kariu T, Ishino T, Yano K, Chinzei Y, Yuda M. CelTOS, a novel malarial protein that mediates transmission to mosquito and vertebrate hosts. Molecular Microbiology. 2006;59(5):1369–1379. [PubMed]
85. Mota MM, Hafalla JCR, Rodriguez A. Migration through host cells activates Plasmodium sporozoites for infection. Nature Medicine Nov. 2002;8(11):1318–1322. [PubMed]
86. Mota MM, Pradel G, Vanderberg JP, et al. Migration of Plasmodium sporozoites through cells before infection. Science. 2001;291(5501):141–144. [PubMed]
87. Ishino T, Yano K, Chinzei Y, Yuda M. Cell-passage activity is required for the malarial parasite to cross the liver sinusoidal cell layer. PLoS Biol. 2004;2(1):E4. [PMC free article] [PubMed]
88. Ishino T, Chinzei Y, Yuda M. A Plasmodium sporozoite protein with a membrane attack complex domain is required for breaching the liver sinusoidal cell layer prior to hepatocyte infection. Cellular Microbiology. 2005;7(2):199–208. [PubMed]
89. Barragan A, Brossier F, Sibley LD. Transepithelial migration of Toxoplasma gondii involves an interaction of intercellular adhesion molecule 1 (ICAM-1) with the parasite adhesin MIC2. Cell Microbiol. 2005;7(4):561–568. [PubMed]