Toxoplasma gondii: Characterization of a Mutant Resistant to 6-Thioxanthine

Pfefferkorn, E. R.; Borotz, Susan E.

doi:10.1006/expr.1994.1099

Cited by 61 publications

(49 citation statements)

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“…To create an ablation construct for precise replacement of the MIC5 open reading frame, we cloned MIC5 5Ј (1.6 kb) and 3Ј (2.5 kb) flanking sequences on either side of the selectable marker, HXGPRTase. The construct was electroporated into a parasite strain lacking expression of HXGPRTase (⌬HX) (18), and parasite clones were selected with mycophenolic acid and xanthine (38). Multiple MIC5 knockout (⌬MIC5) clones from independent transfections were isolated.…”

Section: Resultsmentioning

confidence: 99%

Targeted Deletion of MIC5 Enhances Trimming Proteolysis of Toxoplasma Invasion Proteins

et al. 2006

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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.Members of the phylum Apicomplexa are obligate intracellular parasites that replicate in a variety of cell types. Some, including the human pathogens Plasmodium and Babesia, replicate primarily in the bloodstream, whereas others such as Cryptosporidium, a cause of chronic gastritis among the immune-compromised, and Eimeria, an agricultural parasite, replicate in the intestinal epithelium. Only parasites in the isosporoid coccidian clade, which includes Toxoplasma gondii, the causative agent of toxoplasmosis, replicate in deep tissues (2). Thus, it can be expected that each clade has its own complement of proteins facilitating survival in a specific habitat, along with conserved proteins that play fundamental roles in events common to all apicomplexans.Many invasion studies have been performed in T. gondii, as it is more amenable to in vitro manipulation than other members of the Apicomplexa. Upon contact with a host cell, T. gondii tachyzoites discharge the contents of apically localized microneme (MIC) organelles (13). MIC adhesive proteins contribute to binding host cell receptors, and blocking micronemal secretion dramatically reduces invasion (9). These proteins, which often cluster into multiunit complexes, are transiently deployed to the apical surface during apical attachment and invasion. Transmembrane (TM) MIC proteins, such as MIC2, MIC6, and MIC8, are thought to act as bridging molecules that connect host receptors with the parasite's motility apparatus, the glideosome (37). By translocating MIC-receptor complexes backwards toward the posterior end, the parasite "pulls" itself into the target cell, invaginating the host plasma membrane to form the nascent parasitophorous vacuole (PV). As they treadmill posteriorly,...

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Section: Resultsmentioning

confidence: 99%

Targeted Deletion of MIC5 Enhances Trimming Proteolysis of Toxoplasma Invasion Proteins

et al. 2006

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“…The tachyzoite stage of the RH⌬HXGPRT strain (abbreviated here as RH⌬ [18,29]) was the parent strain used here. It was maintained in vitro by serial passage on monolayers of human foreskin fibroblasts (HFF) at 37°C in 0.5% CO 2 as described previously (20).…”

Section: Methodsmentioning

confidence: 99%

Ionophore-Resistant Mutants of Toxoplasma gondii Reveal Host Cell Permeabilization as an Early Event in Egress

Black

Arrizabalaga

Boothroyd

2000

Molecular and Cellular Biology

131

180

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Toxoplasma gondii is an obligate intracellular pathogen within the phylum Apicomplexa. Invasion and egress by this protozoan parasite are rapid events that are dependent upon parasite motility and appear to be directed by fluctuations in intracellular [Ca 2؉ ]. Treatment of infected host cells with the calcium ionophore A23187 causes the parasites to undergo rapid egress in a process termed ionophore-induced egress (IIE). In contrast, when extracellular parasites are exposed to this ionophore, they quickly lose infectivity (termed ionophoreinduced death [IID]). From among several Iie ؊ mutants described here, two were identified that differ in several attributes, most notably in their resistance to IID. The association between the Iie ؊ and Iid ؊ phenotypes is supported by the observation that two-thirds of mutants selected as Iid ؊ are also Iie ؊ . Characterization of three distinct classes of IIE and IID mutants revealed that the Iie؊ phenotype is due to a defect in a parasite-dependent activity that normally causes infected host cells to be permeabilized just prior to egress. Iie ؊ parasites underwent rapid egress when infected cells were artificially permeabilized by a mild saponin treatment, confirming that this step is deficient in the Iie ؊ mutants. A model is proposed that includes host cell permeabilization as a critical part of the signaling pathway leading to parasite egress. The fact that Iie ؊ mutants are also defective in early stages of the lytic cycle indicates some commonality between these normal processes and IIE.

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“…However, unlike the other selectable markers, HXGPRT is normally expressed by this parasite and thus its expression must be repressed to enable selection for the construct-derived copy. Since it is not an essential gene, the endogenous copy was completely removed in the RH strain, providing a clean background for selection (117,133). Chloramphenicol acetyltransferase is unique among these selectable markers because in addition to its parasiticidal activity, it may be readily used as a reporter (162).…”

Section: Geneticsmentioning

confidence: 99%

Lytic Cycle ofToxoplasma gondii

Black

Boothroyd

2000

Microbiol Mol Biol Rev

450

348

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SUMMARY Toxoplasma gondii is an obligate intracellular pathogen within the phylum Apicomplexa. This protozoan parasite is one of the most widespread, with a broad host range including many birds and mammals and a geographic range that is nearly worldwide. While infection of healthy adults is usually relatively mild, serious disease can result in utero or when the host is immunocompromised. This sophisticated eukaryote has many specialized features that make it well suited to its intracellular lifestyle. In this review, we describe the current knowledge of how the asexual tachyzoite stage of Toxoplasma attaches to, invades, replicates in, and exits the host cell. Since this process is closely analogous to the way in which viruses reproduce, we refer to it as the Toxoplasma “lytic cycle.”

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Toxoplasma gondii: Characterization of a Mutant Resistant to 6-Thioxanthine

Cited by 61 publications

References 0 publications

Targeted Deletion of MIC5 Enhances Trimming Proteolysis of Toxoplasma Invasion Proteins

Targeted Deletion of MIC5 Enhances Trimming Proteolysis of Toxoplasma Invasion Proteins

Ionophore-Resistant Mutants of Toxoplasma gondii Reveal Host Cell Permeabilization as an Early Event in Egress

Lytic Cycle ofToxoplasma gondii

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