Toxoplasma gondii plaque assays revisited: Improvements for ultrastructural and quantitative evaluation of lytic parasite growth

Ufermann, Christoph-Martin; Müller, Florian; Frohnecke, Nora; Laue, Michael; Seeber, Frank

doi:10.1016/j.exppara.2016.12.015

Cited by 9 publications

(9 citation statements)

References 28 publications

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“…3 , B and C ), whereas iΔFd gave rise to only very few and barely visible small plaques when aTc was included in the medium, while iΔFd::Fd retained the capacity to induce plaques. Of note, our initial attempts to interfere with the ferredoxin redox system by using a previously identified single point mutant of the reductase TgFNR, which is enzymatically inactive and shows a tenfold increase in its affinity for TgFd ( 27 ), as a transdominant mutant did not result in an apparent growth defect (see Fig. S2 and associated discussion).…”

Section: Resultsmentioning

confidence: 99%

“…To observe possible ultrastructural changes caused by the knockdown of TgFd, we used correlative light and electron microscopy of parasites within plaques from aTc-treated iΔFd cultures ( 27 ). No apparent morphological changes could be observed when individual tachyzoites within tiny plaques of aTc-treated iΔFd cultures were compared with iΔFd::Fd controls ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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Toxoplasma gondii apicoplast-resident ferredoxin is an essential electron transfer protein for the MEP isoprenoid-biosynthetic pathway

Henkel

Frohnecke

Maus

et al. 2022

Journal of Biological Chemistry

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Toxoplasma gondii apicoplast-resident ferredoxin is an essential electron transfer protein for the MEP isoprenoid-biosynthetic pathway

Henkel

Frohnecke

Maus

et al. 2022

Journal of Biological Chemistry

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“…Toxoplasma gondii proliferation was evaluated in Vero cells by a plaque assay and in HFF determining the tachyzoite yield at 48 h pi. For this assay, Vero cultures grown to confluence in 24-well plates were infected with 5 × 10 4 purified tachyzoites of either TgME49 or TgShSp1 and further maintained at 37°C and 5% CO 2 for 4 days and stained with 0.2% crystal violet (Alfa Aesar, Haverhill, Massachusetts, United States) solution in 2% ethanol (Ufermann et al, 2017). Images were captured using a SMZ1000 binocular loupe (Nikon®, Tokyo, Japan).…”

Section: Methodsmentioning

confidence: 99%

Virulence in Mice of a Toxoplasma gondii Type II Isolate Does Not Correlate With the Outcome of Experimental Infection in Pregnant Sheep

Sánchez-Sánchez

Ferre

Regidor‐Cerrillo

et al. 2019

Front. Cell. Infect. Microbiol.

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Toxoplasma gondii is an apicomplexan parasite that infects almost all warm-blooded animals. Little is known about how the parasite virulence in mice extrapolates to other relevant hosts. In the current study, in vitro phenotype and in vivo behavior in mice and sheep of a type II T. gondii isolate (TgShSp1) were compared with the reference type II T. gondii isolate (TgME49). The results of in vitro assays and the intraperitoneal inoculation of tachyzoites in mice indicated an enhanced virulence for the laboratory isolate, TgME49, compared to the recently obtained TgShSp1 isolate. TgShSp1 proliferated at a slower rate and had delayed lysis plaque formation compared to TgME49, but it formed more cyst-like structures in vitro. No mortality was observed in adult mice after infection with 1–105 tachyzoites intraperitoneally or with 25–2,000 oocysts orally of TgShSp1. In sheep orally challenged with oocysts, TgME49 infection resulted in sporadically higher rectal temperatures and higher parasite load in cotyledons from ewes that gave birth and brain tissues of the respective lambs, but no differences between these two isolates were found on fetal/lamb mortality or lesions and number of T. gondii-positive lambs. The congenital infection after challenge at mid-pregnancy with TgShSp1, measured as offspring mortality and vertical transmission, was different depending on the challenged host. In mice, mortality in 50% of the pups was observed when a dam was challenged with a high oocyst dose (500 TgShSp1 oocysts), whereas in sheep infected with the same dose of oocysts, mortality occurred in all fetuses. Likewise, mortality of 9 and 27% of the pups was observed in mice after infection with 100 and 25 TgShSp1 oocysts, respectively, while in sheep, infection with 50 and 10 TgShSp1 oocysts triggered mortality in 68 and 66% of the fetuses/lambs. Differences in vertical transmission in the surviving offspring were only found with the lower oocyst doses (100% after infection with 10 TgShSp1 oocysts in sheep and only 37% in mice after infection with 25 TgShSp1 oocysts). In conclusion, virulence in mice of T. gondii type II isolates may not be a good indicator to predict the outcome of infection in pregnant sheep.

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“…After plaque development, visually check each well of the 96-well plates under an inverted-phase contrast microscope (such as a Nikon TS100) and look for wells that contain only one plaque (Refer to Ufermann et al ., 2017 for high resolution examples of plaque morphology). Transfer the parasites of positive wells into 24-well plates containing confluent host cell monolayers and grow them until they naturally egress.…”

Section: Methodsmentioning

confidence: 99%

Conditional Knockdown of Proteins Using Auxin-inducible Degron (AID) Fusions in Toxoplasma gondii

Brown¹,

Long²,

Sibley³

2018

BIO-PROTOCOL

107

101

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Toxoplasma gondii is a member of the deadly phylum of protozoan parasites called Apicomplexa. As a model apicomplexan, there is a great wealth of information regarding T. gondii’s 8,000+ protein coding genes including sequence variation, expression, and relative contribution to parasite fitness. However, new tools are needed to functionally investigate hundreds of putative essential protein coding genes. Accordingly, we recently implemented the auxin-inducible degron (AID) system for studying essential proteins in T. gondii. Here we provide a step-by-step protocol for examining protein function in T. gondii using the AID system in a tissue culture setting.

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Toxoplasma gondii plaque assays revisited: Improvements for ultrastructural and quantitative evaluation of lytic parasite growth

Cited by 9 publications

References 28 publications

Toxoplasma gondii apicoplast-resident ferredoxin is an essential electron transfer protein for the MEP isoprenoid-biosynthetic pathway

Toxoplasma gondii apicoplast-resident ferredoxin is an essential electron transfer protein for the MEP isoprenoid-biosynthetic pathway

Virulence in Mice of a Toxoplasma gondii Type II Isolate Does Not Correlate With the Outcome of Experimental Infection in Pregnant Sheep

Conditional Knockdown of Proteins Using Auxin-inducible Degron (AID) Fusions in Toxoplasma gondii

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