Vern B. Carruthers

Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii

Apicomplexan parasites, including Toxoplasma gondii, apically attach to their host cells before invasion. Recent studies have implicated the contents of micronemes, which are small secretory organelles confined to the apical region of the parasite, in the process of host cell attachment. Here, we demonstrate that microneme discharge is regulated by parasite cytoplasmic free Ca2+ and that the micronemal contents, including the MIC2 adhesin, are released through the extreme apical tip of the parasite. Microneme secretion was triggered by Ca2+ ionophores in both the presence and the absence of external Ca2+, while chelation of intracellular Ca2+ prevented release. Mobilization of intracellular calcium with thapsagargin or NH4Cl also triggered microneme secretion, indicating that intracellular calcium stores are sufficient to stimulate release. Following activation of secretion by the Ca2+ ionophore A23187, MIC2 initially occupied the apical surface of the parasite, but was then rapidly treadmilled to the posterior end and released into the culture supernatant. This capping and release of MIC2 by ionophore‐stimulated tachyzoites mimics the redistribution of MIC2 that occurs during attachment and penetration of host cells, and both events are dependent on the actin–myosin cytoskeleton of the parasite. These studies indicate that microneme release is a stimulus‐coupled secretion system responsible for releasing adhesins involved in cell attachment.

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.

Tagging of Endogenous Genes in a Toxoplasma gondii Strain Lacking Ku80

ABSTRACT As with other organisms with a completed genome sequence, opportunities for performing large-scale studies, such as expression and localization, on Toxoplasma gondii are now much more feasible. We present a system for tagging genes endogenously with yellow fluorescent protein (YFP) in a Δku80 strain. Ku80 is involved in DNA strand repair and nonhomologous DNA end joining; previous studies in other organisms have shown that in its absence, random integration is eliminated, allowing the insertion of constructs with homologous sequences into the proper loci. We generated a vector consisting of YFP and a dihydrofolate reductase-thymidylate synthase selectable marker. The YFP is preceded by a ligation-independent cloning (LIC) cassette, which allows the insertion of PCR products containing complementary LIC sequences. We demonstrated that the Δku80 strain is more effective and efficient in integrating the YFP-tagged constructs into the correct locus than wild-type strain RH. We then selected several hypothetical proteins that were identified by a proteomic screen of excreted-secreted antigens and that displayed microarray expression profiles similar to known micronemal proteins, with the thought that these could potentially be new proteins with roles in cell invasion. We localized these hypothetical proteins by YFP fluorescence and showed expression by immunoblotting. Our findings demonstrate that the combination of the Δku80 strain and the pYFP.LIC constructs reduces both the time and cost required to determine localization of a new gene of interest. This should allow the opportunity for performing larger-scale studies of novel T. gondii genes.

Molecular mimicry of a CCR5 binding-domain in the microbial activation of dendritic cells

Toxoplasma gondii releases factors that potently stimulate production of interleukin-12 (IL-12) from dendritic cells (DCs). Purification of this activity showed that cyclophilin-18 (C-18) was its principal component, and antibodies generated against recombinant C-18 inhibited tachyzoite extract–induced synthesis of IL-12. Recombinant C-18 showed high affinity for and triggered cell signaling through CCR5, a chemokine receptor important in parasite-induced IL-12 production by DCs. These findings suggest that the unusual potency of T. gondii in inducing IL-12 from DCs results from its synthesis of a unique chemokine mimic that signals through CCR5. The ability to generate this strong protective response may benefit parasite transmission by preventing the protozoan from overwhelming its intermediate hosts.

Participation of myosin in gliding motility and host cell invasion by Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that actively invades mammalian cells using a unique form of gliding motility that critically depends on actin filaments in the parasite. To determine if parasite motility is driven by a myosin motor, we examined the distribution of myosin and tested the effects of specific inhibitors on gliding and host cell invasion. A single 90 kDa isoform of myosin was detected in parasite lysates using an antisera that recognizes a highly conserved myosin peptide. Myosin was localized in T. gondii beneath the plasma membrane in a circumferential pattern that overlapped with the distribution of actin. The myosin ATPase inhibitor, butanedione monoxime (BDM), reversibly inhibited gliding motility across serum‐coated slides. The myosin light‐chain kinase inhibitor, KT5926, also blocked parasite motility and greatly reduced host cell attachment; however, these effects were primarily caused by its ability to block the secretion of microneme proteins, which are involved in cell attachment. In contrast, while BDM partially reduced cell attachment, it prevented invasion even under conditions in which microneme secretion was not affected, indicating a potential role for myosin in cell entry. Collectively, these results indicate that myosin(s) probably participate(s) in powering gliding motility, a process that is essential for cell invasion by T. gondii.

Molecular characterisation of an expressed sequence tag locus of Toxoplasma gondii encoding the micronemal protein MIC2.

The expressed sequence tag (EST) dataset of Toxoplasma gondii provides a wealth of information towards gene discovery. The complete cDNA and genomic sequence of EST tgc050 locus shows that it contains five copies of the conserved thrombospondin (TSP)-like motif present in a number of molecules with adhesive properties. A conserved region implicated with the adhesive characteristic of another group of proteins including several integrins, is also present in this molecule. The protein encoded by this sequence (rc50) is strongly recognised by monoclonal antibodies to MIC2. Affinity purified anti-rc50 antisera specifically reacted with a single protein of identical molecular mass as MIC2 and exclusively labeled the micronemes of T. gondii by cryo-immunoelectron microscopy. These results demonstrate that c50 encodes for MIC2, a previously characterised microneme protein of T. gondii. The extensive sequence similarity across multiple protein domains provides evidence that the protein encoded by this locus is the homologue to the Etp100 microneme protein of Eimeria tenella.

Rapid Membrane Disruption by a Perforin-Like Protein Facilitates Parasite Exit from Host Cells

Perforin-like proteins are expressed by many bacterial and protozoan pathogens, yet little is known about their function or mode of action. Here, we describe Toxoplasma perforin-like protein 1 (TgPLP1), a secreted perforin-like protein of the intracellular protozoan pathogen Toxoplasma gondii that displays structural features necessary for pore formation. After intracellular growth, TgPLP1-deficient parasites failed to exit normally, resulting in entrapment within host cells. We show that this defect is due to an inability to rapidly permeabilize the parasitophorous vacuole membrane and host plasma membrane during exit. TgPLP1 ablation had little effect on growth in culture but resulted in a reduction greater than five orders of magnitude of acute virulence in mice. Perforin-like proteins from other intracellular pathogens may play a similar role in microbial egress and virulence.

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

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

Host cell invasion by the opportunistic pathogen Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan that infects an astonishing variety of vertebrate hosts including humans. Classified in the phylum Apicomplexa, T. gondii causes an opportunistic disease, toxoplasmosis, in individuals with immune dysfunction and congenital disease in infected infants. Re-emergence of toxoplasmosis as a life-threatening disease in patients with AIDS is anticipated in the wake of emerging multi-drug resistant strains of HIV. In immunodeficient patients, the available evidence suggests that tissue pathology associated with T. gondii infection is due to parasite-directed lytic destruction of individual host cells. The Toxoplasma lytic cycle begins when the parasite actively invades a target cell. In association with invasion, T. gondii sequentially discharges three sets of secretory organelles beginning with the micronemes, which contain adhesive proteins involved in parasite attachment to a host cell. Deployed as protein complexes, several micronemal proteins possess vertebrate-derived adhesive sequences that function in binding receptors on the surface of a target cell. Each protein in these adhesive complexes fulfills a specific role in movement through the secretory pathway, targeting to the micronemes, or adhesion. It is anticipated that these adhesive complexes recognize a variety of host receptors, including some that are expressed on multiple cell types, and that this diversity in host cell receptors contributes to the remarkably broad tissue- and host-range of T. gondii.

ETHANOL AND ACETALDEHYDE ELEVATE INTRACELLULAR CA2+ AND STIMULATE MICRONEME DISCHARGE IN TOXOPLASMA GONDII

One of the first steps in host-cell invasion by the protozoan parasite Toxoplasma gondii occurs when the parasite attaches by its apical end to the target host cell. The contents of apical secretory organelles called micronemes have recently been implicated in parasite apical attachment to host cells. Micronemes are regulated secretory vesicles that discharge in response to elevated parasite intracellular Ca(2+) levels ([Ca2+]i). In the present study we found that ethanol and related compounds produced a dose-dependent stimulation of microneme secretion. In addition, using fluorescence spectroscopy on tachyzoites loaded with the Ca(2+)-sensitive fluorescent dye fura-2, we demonstrated that ethanol stimulated microneme secretion by elevating parasite [Ca2+](i). Furthermore, sequential addition experiments with ethanol and other Ca(2+)-mobilizing drugs showed that ethanol probably elevated parasite [Ca2+](i) by mobilizing Ca(2+) from a thapsigargin-insensitive compartment of neutral pH. Earlier studies have shown that ethanol also elevates [Ca2+](i) in mammalian cells. Thus, because it is genetically tractable, T. gondii might be a convenient model organism for studying the Ca(2+)-elevating effects of alcohol in higher eukaryotes.