Temperature-Sensitive Mutants of Toxoplasma gondii: Pathogenicity and Persistence in Mice

Waldeland, Harald; Pfefferkorn, E. R.; Frenkel, J. K.

doi:10.2307/3281293

Cited by 44 publications

(17 citation statements)

References 8 publications

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“…To determine the molecular basis of dinitroaniline action, we generated oryzalin-resistant lines by using the chemical mutagen N-nitroso-N-ethylurea (Waldeland et al, 1983;Pfefferkorn, 1984;Dobrowolski and Sibley, 1996). Because Toxoplasma is haploid, genetic changes that confer resistance are readily detected, including mutations that are recessive in diploid organisms.…”

Section: Oryzalin-resistant Tachyzoites Have Diverse Phenotypesmentioning

confidence: 99%

Dinitroanilines Bind α-Tubulin to Disrupt Microtubules

Morrissette

Mitra

Sept

et al. 2004

MBoC

137

153

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Protozoan parasites are remarkably sensitive to dinitroanilines such as oryzalin, which disrupt plant but not animal microtubules. To explore the basis of dinitroaniline action, we isolated 49 independent resistant Toxoplasma gondii lines after chemical mutagenesis. All 23 of the lines that we examined harbored single point mutations in ␣-tubulin. These point mutations were sufficient to confer resistance when transfected into wild-type parasites. Several mutations were in the M or N loops, which coordinate protofilament interactions in the microtubule, but most of the mutations were in the core of ␣-tubulin. Docking studies predict that oryzalin binds with an average affinity of 23 nM to a site located beneath the N loop of Toxoplasma ␣-tubulin. This binding site included residues that were mutated in several resistant lines. Moreover, parallel analysis of Bos taurus ␣-tubulin indicated that oryzalin did not interact with this site and had a significantly decreased, nonspecific affinity for vertebrate ␣-tubulin. We propose that the dinitroanilines act through a novel mechanism, by disrupting M-N loop contacts. These compounds also represent the first class of drugs that act on ␣-tubulin function. INTRODUCTIONMicrotubules are polymers constructed from ␣-␤-tubulin heterodimers (Downing and Nogales, 1998a,b). These structures are rapidly assembled and disassembled to create essential components of eukaryotic cells, such as spindles and flagella. The dynamic nature of microtubules makes them susceptible to pharmacological agents. Microtubule-disrupting and microtubule-stabilizing drugs have provided great insight into tubulin and microtubule function; they also have tremendous practical use. Compounds that perturb microtubule dynamics are currently some of the most effective drugs to treat medical conditions, including cancer, gout, and helminth infection (Jordan et al., 1998). Dinitroanilines (oryzalin, ethafluralin, and trifluralin) disrupt the microtubules of plants, ranging from the single-celled alga Chlamydomonas reinhardtii to higher plants such as the monocot Eleusine indica (James et al., 1993;Anthony et al., 1998;Zeng and Baird, 1999). Dinitroanilines also disrupt the microtubules of protozoa, including both free-living species such as Tetrahymena and protozoan parasites such as Trypanosoma spp., Leishmania spp., Entamoeba spp., Plasmodium falciparum, Cryptosporidium parvum, and Toxoplasma gondii (Chan and Fong, 1990;Chan et al., 1991;Gu et al., 1995;Edlind et al., 1996;Stokkermans et al., 1996;Armson et al., 1999; Makioka et al., 2000a,b;Traub-Cseko et al., 2001). Remarkably, the activity of dinitroanilines is restricted to plants and protozoa; these compounds are ineffective against vertebrate or fungal microtubules (Chan and Fong, 1990;Hugdahl and Morejohn, 1993;Murthy et al., 1994;Edlind et al., 1996).T. gondii is a member of the Apicomplexa, a phylum of parasites that includes several medically and agriculturally significant pathogens (Black and Boothroyd, 2000). Apicomplexans are obligate intracell...

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Section: Oryzalin-resistant Tachyzoites Have Diverse Phenotypesmentioning

confidence: 99%

Dinitroanilines Bind α-Tubulin to Disrupt Microtubules

Morrissette

Mitra

Sept

et al. 2004

MBoC

137

153

View full text Add to dashboard Cite

show abstract

“…The temperature-sensitive mutant ts-4 derived from the RH strain (13,14) was passaged every 3-5 days in tissue culture in human foreskin fibroblasts. Me49, a less virulent (type II) (15) strain that results in chronic infection with encysted bradyzoites, was passaged every 3-5 mo in Swiss-Webster mice as previously described (12).…”

Section: T Gondiimentioning

confidence: 99%

In Vitro Correlates ofLd-Restricted Resistance to Toxoplasmic Encephalitis and Their Critical Dependence on Parasite Strain

Johnson

Roberts

Pope

et al. 2002

The Journal of Immunology

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Resistance to murine toxoplasmic encephalitis has been precisely and definitively mapped to the Ld class I gene. Consistent with this, CD8+ T cells can adoptively transfer resistance to toxoplasmic encephalitis. However, cytotoxic CD8+ T cells, capable of killing class I-matched, infected target cells, are generated during the course of Toxoplasma gondii infection even in mice lacking the Ld gene. Ld-restricted killing could not be demonstrated, and the functional correlate of the Ld gene has therefore remained elusive. Herein, Ld-restricted killing of T. gondii-infected target cells is demonstrated for the first time. Ld-restricted killing is critically dependent on the strain of T. gondii and is observed with all the derivatives of type II strains tested, but not with a type I strain. These results have important implications for vaccine development.

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“…This mutant was originally selected for temperature sensitivity in vitro (14) and has been maintained by serial passage for over 25 years as an attenuated parasite line. Importantly, ts-4 infections do not persist in mice longer than 8 wk (13,42), although it is still unclear whether the temperature-sensitivity phenotype is directly responsible for this lack of virulence. Since ts-4 induces solid protection against challenge infection with RH, it was a logical choice for a vehicle to vaccinate against other intracellular pathogens.…”

Section: Discussionmentioning

confidence: 99%

“…The temperature-sensitive ts-4 mutant strain of RH, in contrast, is avirulent in the experimental murine model. While the injection of only one wild-type RH tachyzoite results in death within 12 days, mice inoculated with up to 10 7 ts-4 tachyzoites survive and do not show acute disease symptoms (13,14). Importantly, mice inoculated with ts-4 tachyzoites develop complete protection against lethal challenge with RH (14 -16).…”

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confidence: 99%

Recombinant Attenuated Toxoplasma gondii Expressing the Plasmodium yoelii Circumsporozoite Protein Provides Highly Effective Priming for CD8+ T Cell-Dependent Protective Immunity Against Malaria

Charest

Sedegah

Yap

et al. 2000

The Journal of Immunology

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The protozoan parasite Toxoplasma gondii elicits strong cell-mediated immunity against itself as well as nonspecific resistance against other pathogens and tumors. For this reason, we asked whether recombinant Toxoplasma could be utilized as an effective vaccine vehicle for inducing immunity against heterologous microbial infections. The circumsporozoite protein (PyCSP) of Plasmodium yoelii was engineered into a T. gondii temperature-sensitive strain (ts-4), a mutant that induces complete protection against virulent Toxoplasma challenge. When administered to mice in a single dose, a recombinant ts-4 (CSC3) that both secretes and expresses surface PyCSP induced strong anti-CSP Ab responses, with an isotype distribution pattern similar to that stimulated by the T. gondii carrier. When challenged with P. yoelii sporozoites during the first month after CSC3 vaccination, these animals displayed substantial levels of nonspecific resistance attributable entirely to the T. gondii carrier. Nevertheless, after the nonspecific protection had waned, high levels (up to 79%) of specific immunity against sporozoite challenge were achieved by boosting the animals with recombinant vaccinia virus expressing PyCSP. These CSC3-primed PyCSP-vaccinia-boosted mice displayed high frequencies of splenic PyCSP-specific IFN-γ-producing cells, as well as CD8+ T cell-dependent cytolytic activity. In vivo depletion of CD8+ lymphocytes at the time of challenge completely ablated protective immunity in the T. gondii-primed/vaccinia-boosted animals, while neutralization of IFN-γ or IL-12 caused a partial but significant reduction in resistance. Together these findings establish the efficacy of recombinant attenuated Toxoplasma as a vaccine vehicle for priming CD8+-dependent cell-mediated immunity.

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Temperature-Sensitive Mutants of Toxoplasma gondii: Pathogenicity and Persistence in Mice

Cited by 44 publications

References 8 publications

Dinitroanilines Bind α-Tubulin to Disrupt Microtubules

Dinitroanilines Bind α-Tubulin to Disrupt Microtubules

In Vitro Correlates ofLd-Restricted Resistance to Toxoplasmic Encephalitis and Their Critical Dependence on Parasite Strain

Recombinant Attenuated Toxoplasma gondii Expressing the Plasmodium yoelii Circumsporozoite Protein Provides Highly Effective Priming for CD8+ T Cell-Dependent Protective Immunity Against Malaria

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