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

Brydges, Susannah; Sherman, Gale D.; Nockemann, Susanne; Loyens, Anne; Däubener, Walter; Dubremetz, Jean François; Carruthers, Vern B.

doi:10.1016/s0166-6851(00)00296-6

Cited by 64 publications

(50 citation statements)

References 45 publications

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“…The contents of the micronemes are secreted constitutively at a low basal rate, and secretion is up-regulated in response to increases in intracellular calcium (13,19). The effect of the small molecules on constitutive microneme secretion was determined by Western blotting, using the proteolytically processed form of the soluble microneme protein, MIC5 (20), as a marker. Release of soluble ␤-galactosidase from the parasites was used to distinguish secretion from nonspecific parasite lysis (13).…”

Section: Resultsmentioning

confidence: 99%

A small-molecule approach to studying invasive mechanisms of Toxoplasma gondii

Carey

Westwood

Mitchison

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

130

163

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Toxoplasma gondii is the most common protozoan parasite of humans. Infection with T. gondii can lead to life-threatening disease as a result of repeated cycles of host cell invasion, parasite replication, and host cell lysis. Relatively little is known about the invasive mechanisms of T. gondii and related parasites within the Phylum Apicomplexa (including Plasmodium spp., the causative agents of malaria), due to difficulties associated with studying genes essential to invasion in haploid obligate intracellular organisms. To circumvent this problem, we have developed a highthroughput microscope-based assay, which we have used to screen a collection of 12,160 structurally diverse small molecules for inhibitors of T. gondii invasion. A total of 24 noncytotoxic invasion inhibitors were identified. Secondary assays demonstrated that different inhibitors perturb different aspects of invasion, including gliding motility, secretion of host cell adhesins from apical organelles (the micronemes), and extension of a unique tubulinbased structure at the anterior of the parasite (the conoid). Unexpectedly, the screen also identified six small molecules that dramatically enhance invasion, gliding motility, and microneme secretion. The small molecules identified here reveal a previously unrecognized complexity in the control of parasite motility and microneme secretion, and they constitute a set of useful probes for dissecting the invasive mechanisms of T. gondii and related parasites. Small-molecule-based approaches provide a powerful means to address experimentally challenging problems in host-pathogen interaction, while simultaneously identifying new potential targets for drug development. N early one-third of all deaths in the world today are caused by infectious disease. The development of new preventative and therapeutic strategies relies on an improved understanding of the interaction between pathogens and their hosts. In many pathogenic systems, this presents a formidable experimental challenge, because standard genetic tools are either rudimentary or unavailable. Biochemical, genomic, and other approaches exist, but these lack the assumption-free power of a genetic screen.An alternative nongenetic approach to studying mechanisms of host-pathogen interaction involves screening large structurally diverse collections of small molecules for those that disrupt the interaction. Once identified, the small molecules (or their derivatives) are used to determine the cellular components that function in the process (reviewed in refs. 1-3). The approach relies on the demonstrated ability of many small molecules to interact specifically with their targets (e.g., ref. 4). As with classical forward genetic screens, the approach is assumptionfree: by sampling large unbiased collections of structurally diverse small molecules, the screen ''selects'' structures that perturb the process under study. Such phenotype-based highthroughput screening has recently gained momentum in the academic setting due to technological advances and the av...

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Section: Resultsmentioning

confidence: 99%

A small-molecule approach to studying invasive mechanisms of Toxoplasma gondii

Carey

Westwood

Mitchison

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

130

163

View full text Add to dashboard Cite

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“…BFA is effective at blocking transport in mammalian cells at concentrations of 50-100 ng/ml, and has generally been used at concentrations of 5-10 µg/ml for studies in T. gondii (Brydges et al, 2000;Soldati et al, 1998;Stedman et al, 2003), although 1 µg/ml has been shown to prevent secretion of GRA1 (Coppens et al, 1999). Preliminary studies showed that T. gondii were able to proliferate following treatment with 1 µg/ml BFA for 4 hours, but not if the concentration was raised to 10 µg/ml (not shown).…”

Section: Resultsmentioning

confidence: 99%

Dissection of brefeldin A-sensitive and -insensitive steps in apicoplast protein targeting

DeRocher

Gilbert

Feagin

et al. 2005

Journal of Cell Science

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The apicoplast is a relict plastid found in many apicomplexans, including the pathogens Toxoplasma gondii and Plasmodium falciparum. Nucleus-encoded apicoplast proteins enter the ER, and after cleavage of the signal sequence, are routed to the apicoplast by virtue of a transit peptide, which is subsequently removed. To assess the mechanisms of localization we examined stable transfectants of T. gondii for the localization and processing of various GFP fusion proteins. GFP fusions bearing apicoplast targeting sequences targeted efficiently to the plastid, with no retention in the ER, even when an ER retention/retrieval sequence was added. Incubation with brefeldin A, which blocks ER-to-Golgi trafficking by inhibiting a GTP exchange factor required for retrograde trafficking, blocked the processing of the protein. Surprisingly, it did not affect the immunofluorescence pattern. To avoid the potentially misleading presence of pre-existing GFP fusion protein in the apicoplast, we used a ligand-regulated aggregation system to arrest the GFP fusion protein in the ER prior to trafficking. Upon addition of ligand to promote disaggregation, the fusion protein targeted to the plastid, even in the presence of brefeldin A. Ligand release at 15°C, which blocks trafficking of Golgirouted proteins, also allowed significant localization to the plastid. Our data indicate that apicoplast proteins can localize to the region of the plastid when Golgi trafficking is inhibited, but suggest that some steps in import or maturation of the proteins may require a brefeldin A-sensitive GTP exchange factor.

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“…Mut⌬C was localized to the apical region of the parasite as demonstrated by its colocalization with microneme protein five (MIC5) (Fig. 1A) (22). To ensure that the apical pattern of mut⌬C was due to localization within micronemes and not another apical vesicle, cryoimmunoelectron microscopy was employed.…”

Section: Fig 1 Mut⌬c Is Localized To the Micronemesmentioning

confidence: 99%

The Toxoplasma Proteins MIC2 and M2AP Form a Hexameric Complex Necessary for Intracellular Survival

Jewett

Sibley

2004

Journal of Biological Chemistry

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Toxoplasma gondii parasites gain entry into host cells through a process that depends on apically stored adhesins that are strategically released during invasion. One of these adhesins, microneme protein 2 (MIC2), is a type one transmembrane protein that binds to an accessory protein known as MIC2-associated protein (M2AP). Together the MIC2⅐M2AP complex participates in host cell attachment and invasion. The short cytoplasmic Cdomain of MIC2 is implicated in protein trafficking and mediating an association with the parasite cytoskeleton. To define the role of the cytoplasmic domain of MIC2, proteins lacking the C-domain were expressed in transgenic T. gondii. Surprisingly, protein trafficking and secretion were not affected. We hypothesized that mutant mic2 lacking the C-domain might be escorted to the micronemes by association with endogenous wild-type MIC2 possessing functional transmembrane and cytoplasmic domains. To investigate this interaction, native blue gels and gel filtration were employed to identify a stable macromolecular MIC2⅐M2AP complex of ϳ450 kDa. Our findings reveal that MIC2 and M2AP proteins form stable hexamers consisting of three ␣␤ dimers. Resolution of this complex has implications for how MIC2⅐M2AP associates with host cell receptors and the cytoskeleton to facilitate parasite motility and invasion.Apicomplexan parasites actively gain entry into host cells by a unique mechanism that depends on the cohesive interaction of cytoskeletal proteins and surface adhesins (1). Host cell invasion is fundamental to diseases caused by this phylum of pathogenic organisms that includes Plasmodium falciparum, the etiological agent of severe malaria and Toxoplasma gondii, the causative agent of toxoplasmosis. Tachyzoites (Toxoplasma) and sporozoites (Plasmodium) employ similar adhesins for active entry into host cells. These adhesins define a family of thrombospondin-related anonymous proteins (TRAPs) 1 (2, 3). TRAP family members are type 1 transmembrane proteins that are comprised of adhesive N-terminal domains that harbor integrin-like A-domains and at least one thrombospondin like domain and a short C-terminal cytoplasmic domain (C-domain) (2, 3). Toxoplasma gondii contains a single TRAP family member called MIC2, which is expressed by all of the invasive stages of the parasite. MIC2 is stored in the apically located microneme secretory organelles. Following host cell contact, microneme vesicles fuse with the proximal plasma membrane depositing MIC2 on the surface of the parasite (4 -6). Images of T. gondii captured at various stages of invasion indicate that MIC2 is translocated to the posterior end of the parasite during the rapid process of entry (7). Ultimately, MIC2 is removed from the parasite surface by a protease that cleaves the MIC2 protein within the transmembrane domain (8, 9). Amino acids within and just external to the transmembrane domain are implicated in this processing event (9, 10). Recently, a Drosophila rhomboid protease was found to cleave a chimeric substrate containing the M...

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

Cited by 64 publications

References 45 publications

A small-molecule approach to studying invasive mechanisms of Toxoplasma gondii

A small-molecule approach to studying invasive mechanisms of Toxoplasma gondii

Dissection of brefeldin A-sensitive and -insensitive steps in apicoplast protein targeting

The Toxoplasma Proteins MIC2 and M2AP Form a Hexameric Complex Necessary for Intracellular Survival

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