[Show abstract][Hide abstract] ABSTRACT: Accurate sorting of proteins to the three types of secretory granules in Toxoplasma gondii is crucial for successful cell invasion by this obligate intracellular parasite. As in other eukaryotic systems, propeptide sequences are a common yet poorly understood feature of proteins destined for regulated secretion, which for Toxoplasma occurs through two distinct invasion organelles, rhoptries and micronemes. Microneme discharge during parasite apical attachment plays a pivotal role in cell invasion by delivering adhesive proteins for host receptor engagement.
We show here that the small micronemal proprotein MIC5 (microneme protein-5) undergoes proteolytic maturation at a site beyond the Golgi, and only the processed form of MIC5 is secreted via the micronemes. Proper cleavage of the MIC5 propeptide relies on an arginine residue in the P1' position, although P1' mutants are still cleaved to a lesser extent at an alternative site downstream of the primary site. Nonetheless, this aberrantly cleaved species still correctly traffics to the micronemes, indicating that correct cleavage is not necessary for micronemal targeting. In contrast, a deletion mutant lacking the propeptide was retained within the secretory system, principally in the ER (endoplasmic reticulum). The MIC5 propeptide also supported correct trafficking when exchanged for the M2AP propeptide, which was recently shown to also be required for micronemal trafficking of the TgMIC2 (T. gondii MIC2)-M2AP complex [Harper, Huynh, Coppens, Parussini, Moreno and Carruthers (2006) Mol. Biol. Cell 17, 4551-4563].
Our results illuminate common and unique features of micronemal propeptides in their role as trafficking facilitators.
Biology of the Cell 05/2008; 100(4):253-64. DOI:10.1042/BC20070076 · 3.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Limited proteolysis of proteins transiently expressed on the surface of the opportunistic pathogen Toxoplasma gondii accompanies cell invasion and facilitates parasite migration across cell barriers during infection. However, little is known about what factors influence this specialized proteolysis or how these proteolytic events are regulated. Here we show that genetic ablation of the micronemal protein MIC5 enhances the normal proteolytic processing of several micronemal proteins secreted by Toxoplasma tachyzoites. Restoring MIC5 expression by genetic complementation reversed this phenotype, as did treatment with the protease inhibitor ALLN, which was previously shown to block the activity of a hypothetical parasite surface protease called MPP2. We show that, despite its lack of obvious membrane association signals, MIC5 occupies the parasite surface during invasion in the vicinity of the proteins affected by enhanced processing. Proteolysis of other secretory proteins, including GRA1, was also enhanced in MIC5 knockout parasites, indicating that the phenotype is not strictly limited to proteins derived from micronemes. Together, our findings suggest that MIC5 either directly regulates MPP2 activity or it influences MPP2's ability to access substrate cleavage sites on the parasite surface.
[Show abstract][Hide abstract] ABSTRACT: Propeptides regulate protein function and trafficking in many eukaryotic systems and have emerged as important features of regulated secretory proteins in parasites of the phylum Apicomplexa. Regulated protein secretion from micronemes and host cell invasion are inextricably linked and essential processes for the apicomplexan parasite Toxoplasma gondii. TgM2AP is a propeptide-containing microneme protein found in a heterohexameric complex with the microneme protein TgMIC2, a protein that has a demonstrated fundamental role in gliding motility and invasion. TgM2AP function is also central to these processes, because disruption of TgM2AP (m2apKO) results in secretory retention of TgMIC2, leading to reduced TgMIC2 secretion from the micronemes and impaired invasion. Because the TgM2AP propeptide is predicted to be processed in an intracellular site near where TgMIC2 is retained in m2apKO parasites, we hypothesized that the propeptide and its proteolytic removal influence trafficking and secretion of the complex. We found that proTgM2AP traffics through endosomal compartments and that deletion of the propeptide leads to defective trafficking of the complex within or near this site, resulting in aberrant processing and decreased secretion of TgMIC2, impaired invasion, and reduced virulence in vivo, mirroring the phenotypes observed in m2apKO parasites. In contrast, mutation of several cleavage site residues resulted in normal localization, but it affected the stability and secretion of the complex from the micronemes. Therefore, the propeptide and its cleavage site influence distinct aspects of TgMIC2-M2AP function, with both impacting the outcome of infection.
Molecular Biology of the Cell 11/2006; 17(10):4551-63. DOI:10.1091/mbc.E06-01-0064 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Toxoplasma gondii is an apicomplexan parasite capable of infecting a broad host range including humans. The tachyzoite lytic cycle begins with active invasion of host cells involving the release of adhesive proteins from apical secretory organelles called micronemes. A protein complex consisting of the transmembrane adhesin MIC2 and a tightly associated partner, M2AP, is abundantly released from the micronemes. Similar to many proteins in a regulated secretory pathway, T. gondii proteins destined for micronemes and rhoptries (another secretory organelle associated with invasion) undergo proteolytic maturation. M2AP contains a propeptide that is removed in a post-Golgi compartment. By expressing an M2AP propeptide deletion mutant in the M2AP knockout background, we show that the propeptide is required for the MIC2-M2AP complex to exit from the early endosome. Although a cleavage-resistant M2AP mutant was able to efficiently reach the micronemes, it was unable to rapidly mobilize from the micronemes to the parasite surface. Strikingly, both mutants were unable to support normal parasite invasion and were partially attenuated in virulence to a degree that is indistinguishable from M2AP knockout parasites. Conditional expression of MIC2 showed that it is also required for correct M2AP sorting to the micronemes. These parasites were severely impaired in invasion efficiency. They switched almost exclusively to a non-productive circular gliding motility and were incapable of establishing an infection in mice when inoculated at a normally lethal dose. These findings underscore the importance of correct trafficking of invasion-related proteins. Our results also serve as a basis for future studies aimed at defining the branch points of protein sorting in T. gondii and at a deeper understanding of the precise roles of M2AP propeptide and MIC2 targeting motifs in MIC protein trafficking.
Parasitology Research 05/2006; 98(5):389-95. DOI:10.1007/s00436-005-0062-2 · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Host cell invasion is a key step in the life cycle of the intracellular parasite Toxoplasma gondii, the causative agent of toxoplasmosis. Attachment and invasion by this parasite is dependent on secretion of proteins from the micronemes, cigar-shaped organelles found in the apical end of the parasite. Although many of these proteins contain adhesive motifs suggestive of a role in parasite attachment, a growing subset of microneme proteins (MICs) do not possess adhesive sequences implying that they have alternative roles. We have identified a novel 16 kDa microneme protein, TgMIC11, that is conserved among several coccidian parasites. As it traffics through the secretory system, TgMIC11 is modified by two successive proteolytic events to remove an internal propeptide, resulting in the mature protein that consists of an alpha-chain and beta-chain tethered by a single disulfide bond. Dual staining immunofluorescence confirmed that TgMIC11 localises to the apical micronemes and, like other micronemal proteins, it is also secreted in a calcium dependent manner. This is the first microneme protein characterised to date in the phylum Apicomplexa that possesses this unique structure and undergoes maturation by removal of an internal propeptide.
International Journal for Parasitology 09/2004; 34(9):1047-58. DOI:10.1016/j.ijpara.2004.05.006 · 3.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis in humans and animals. To invade host cells, T. gondii deploys the contents of its apically oriented secretory organelles that include the micronemes. Contained within the micronemes are proteins that possess adhesive motifs resembling those found in mammalian proteins. The micronemal protein MIC2 is a member of the thrombospondin-related anonymous protein (TRAP) family of adhesive proteins, which characteristically feature at least one integrin-like A-domain. Because of its strict conservation within the family, we sought to define the role of this domain by testing the adhesive properties of recombinant MIC2 A-domain fusion proteins. Since MIC2 is found as a multimeric species in parasite lysate, we also wanted to test whether recombinant MIC2 A-domain bound to its substrate in a multimeric state. In vitro assays of binding to several different potential receptors revealed that the MIC2 A-domain binds specifically to heparin, a ubiquitous sulfated proteoglycan found in the extracellular matrix (ECM). Additional studies demonstrated that this binding is not dependent on the MIDAS site, a well-conserved divalent cation-binding motif that the MIC2 A-domain shares with its mammalian counterparts. The recombinant MIC2 A-domain bound to heparin as a high molecular weight species, as did MIC2 from parasite lysate, indicating that the recombinant A-domain mimics the binding of native MIC2. Multimerization of MIC2 may increase the number of interactions with host cell receptors, thereby forming a multivalent adhesive junction during parasite entry.
[Show abstract][Hide abstract] ABSTRACT: 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.
The EMBO Journal 05/2003; 22(9):2082-90. DOI:10.1093/emboj/cdg217 · 10.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 Eimeriaspecies, 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
[Show abstract][Hide abstract] ABSTRACT: Hoff, E. F., Cook, S. H., Sherman, G. D., Harper, J. M., Ferguson, D. J. P., Dubremetz, J. F., and Carruthers, V. B. 2001. Toxoplasma gondii: Molecular cloning and characterization of a novel 18-kDa secretory antigen, TgMIC10. Experimental Parasitology, 97, 77-88. During host cell invasion, Toxoplasma gondii secretes proteins from specialized organelles (micronemes and rhoptries) located at the apical end of the parasite. The contents of the micronemes appear to be crucial to T. gondii invasion, as inhibition of microneme secretion prevents parasite entry into host cells. Here we describe a new T. gondii microneme protein, TgMIC10. Molecular characterization of a full-length TgMIC10 cDNA revealed that TgMIC10 lacks homology to any previously characterized proteins, although a homologue, NcMIC10, was identified in a closely related parasite, Neospora caninum. TgMIC10 has an unusually long secretory leader sequence of 58 amino acids; the mature TgMIC10 is 18 kDa, possesses nine diglutamic acid repeats and an imperfect repeat sequence (RK(R/Y)HEEL), and is entirely devoid of cysteines. Antibodies raised against recombinant TgMIC10 recognized the native TgMIC10 and localized the protein to the micronemes in indirect immunofluorescence and immunoEM experiments. Comparison of immunofluorescence images indicates that TgMIC10 expression is higher in T. gondii tachyzoites, which are responsible for active infection, than in bradyzoites, which are responsible for latent infection.