Optimized expression of green fluorescent protein in Toxoplasma gondii using thermostable green fluorescent protein mutants

Kim, Kami; Eaton, Michael; Schubert, William; Wu, Song; Tang, Justin

doi:10.1016/s0166-6851(01)00212-2

Cited by 57 publications

(54 citation statements)

References 10 publications

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“…Growth competition assays indicate that the derived lines have increased fitness relative to the parental strains: We have exploited an established GFP-expressing T. gondii line to assess the relative fitness of the F52Y and G142S parasite lines and the derived progeny (Kim et al 2001). Both the GFP-expressing line and the tubulin mutant lines are derived from RH strain T. gondii.…”

Section: Resultsmentioning

confidence: 99%

“…Competition assays: T 25 flasks with confluent HFF cells were inoculated with a 1:1 ratio of RH-strain-derived lines described here and wild-type (RH strain) parasites expressing cytosolic GFP (1 3 10 7 parasites from each line) (Kim et al 2001). After host monolayer lysis, a new T 25 flask with confluent HFF cells was inoculated with 1.0 ml of the lysed culture and the remaining material (5 ml) was used for flow cytometry analysis.…”

Section: Methodsmentioning

confidence: 99%

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Secondary Mutations Correct Fitness Defects inToxoplasma gondiiWith Dinitroaniline Resistance Mutations

I-Hsing

Tran

et al. 2008

Genetics

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Dinitroanilines (oryzalin, trifluralin, ethafluralin) disrupt microtubules in protozoa but not in vertebrate cells, causing selective death of intracellular Toxoplasma gondii parasites without affecting host cells. Parasites containing a1-tubulin point mutations are dinitroaniline resistant but show increased rates of aberrant replication relative to wild-type parasites. T. gondii parasites bearing the F52Y mutation were previously demonstrated to spontaneously acquire two intragenic mutations that decrease both resistance levels and replication defects. Parasites bearing the G142S mutation are largely dependent on oryzalin for viable growth in culture. We isolated 46 T. gondii lines that have suppressed microtubule defects associated with the G142S or the F52Y mutations by acquiring secondary mutations. These compensatory mutations were a1-tubulin pseudorevertants or extragenic suppressors (the majority alter the b1-tubulin gene). Many secondary mutations were located in tubulin domains that suggest that they function by destabilizing microtubules. Most strikingly, we identified seven novel mutations that localize to an eight-amino-acid insert that stabilizes the a1-tubulin M loop, including one (P364R) that acts as a compensatory mutation in both F52Y and G142S lines. These lines have reduced dinitroaniline resistance but most perform better than parental lines in competition assays, indicating that there is a trade-off between resistance and replication fitness.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Secondary Mutations Correct Fitness Defects inToxoplasma gondiiWith Dinitroaniline Resistance Mutations

I-Hsing

Tran

et al. 2008

Genetics

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“…Parasites were harvested and purified by filtration using 3-m polycarbonate filters as previously described (31). Parasite strains used in this study include RH p30GFP-HDEL (16), ROP-GFP (47), and RH GFP 5S65T (21).…”

Section: Methodsmentioning

confidence: 99%

A Novel Actin-Related Protein Is Associated with Daughter Cell Formation in Toxoplasma gondii

2008

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Cell division in Toxoplasma gondii occurs by an unusual budding mechanism termed endodyogeny, during which twin daughters are formed within the body of the mother cell. Cytokinesis begins with the coordinated assembly of the inner membrane complex (IMC), which surrounds the growing daughter cells. The IMC is compiled of both flattened membrane cisternae and subpellicular filaments composed of articulin-like proteins attached to underlying singlet microtubules. While proteins that comprise the elongating IMC have been described, little is known about its initial formation. Using Toxoplasma as a model system, we demonstrate that actin-like protein 1 (ALP1) is partially redistributed to the IMC at early stages in its formation. Immunoelectron microscopy localized ALP1 to a discrete region of the nuclear envelope, on transport vesicles, and on the nascent IMC of the daughter cells prior to the arrival of proteins such as IMC-1. The overexpression of ALP1 under the control of a strong constitutive promoter disrupted the formation of the daughter cell IMC, leading to delayed growth and defects in nuclear and apicoplast segregation. Collectively, these data suggest that ALP1 participates in the formation of daughter cell membranes during cell division in apicomplexan parasites.

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“…Upstream (promoter) sequences from the TZ-specific GRA1 (pGRA1) (30) and from the BZ-specific SRS9 (pSRS9) were used to express transgenic SRS9 and SAG1 in the TZ and BZ stage, respectively. The SRS9 promoter region (ϳ1.5 kb) was PCR cloned from the Pru strain genomic DNA using the following primers: 5Ј-GGGGAAGCTTTGTCACCGGTTCGGTGCACT-3Ј and 5Ј-GCCCATGCATTGTGTCGACCCGTGTGCACG-3Ј.…”

Section: Generation Of Srs9-constitutive (Srs9 Cmentioning

confidence: 99%

Stage-Specific Expression of Surface Antigens by Toxoplasma gondii as a Mechanism to Facilitate Parasite Persistence

Kim

Boothroyd

2005

The Journal of Immunology

103

106

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Toxoplasma persists in the face of a functional immune system. This success critically depends on the ability of parasites to activate a strong adaptive immune response during acute infection with tachyzoites that eliminates most of the parasites and to undergo stage conversion to bradyzoites that encyst and persist predominantly in the brain. A dramatic change in antigenic composition occurs during stage conversion, such that tachyzoites and bradyzoites express closely related but antigenically distinct sets of surface Ags belonging to the surface Ag 1 (SAG1)-related sequence (SRS) family. To test the contribution of this antigenic switch to parasite persistence, we engineered parasites to constitutively express the normally bradyzoite-specific SRS9 (SRS9c) mutants and tachyzoite-specific SAG1 (SAG1c) mutants. SRS9c but not wild-type parasites elicited a SRS9-specific immune response marked by IFN-γ production, suggesting that stage-specificity of SRS Ags determines their immunogenicity in infection. The induction of a SRS9-specific immune response correlated with a continual decrease in the number of SRS9c cysts persisting in the brain. In contrast, SAG1c mutants produced reduced brain cyst loads early in chronic infection, but these substantially increased over time accompanying a hyperproduction of IFN-γ, TNF-α, and IL-10, and severe encephalitis. We conclude that stage-specific expression of SRS Ags is among the key mechanisms by which optimal parasite persistency is established and maintained.

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Optimized expression of green fluorescent protein in Toxoplasma gondii using thermostable green fluorescent protein mutants

Cited by 57 publications

References 10 publications

Secondary Mutations Correct Fitness Defects inToxoplasma gondiiWith Dinitroaniline Resistance Mutations

Secondary Mutations Correct Fitness Defects inToxoplasma gondiiWith Dinitroaniline Resistance Mutations

A Novel Actin-Related Protein Is Associated with Daughter Cell Formation in Toxoplasma gondii

Stage-Specific Expression of Surface Antigens by Toxoplasma gondii as a Mechanism to Facilitate Parasite Persistence

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