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A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants
Published on: February 8, 2012
Development of a screen to dissect Toxoplasma gondii egress
Keith P Eidell1, Thomas Burke, Marc-Jan Gubbels
1Department of Biology, Boston College, 140 Commonwealth Avenue, Chestnut Hill, MA 02467, USA.
Abstract:
Toxoplasma gondii egress from the host cell during the lytic part of its life cycle is increasingly appreciated as a process where complex signaling mediates the parasite's response to a variety of internal and external conditions. Although several in vitro as well as physiological triggers have been identified, the molecular nature of these signaling pathways is largely unexplored. To facilitate a more comprehensive analysis of the underlying mechanism we designed a screening procedure to enrich for phenotypes with defects in induced egress. The procedure is based on in vitro induced egress and the efficient separation of intracellular from extracellular parasites. Attachment and fast reinvasion of egressed parasites are prevented by the addition of glycans, whereas PDTC is included to specifically kill the egressed, extracellular parasites. Two available mutants were used to assess the power of the screen; a temperature sensitive mutant, F-P2, with a conditionally lethal, reversible egress defect, and a mutant wherein the perforin PLP1 is knocked out displaying a constitutive, delayed egress defect. We show that mutant F-P2 can be routinely enriched over 1000-fold from a wild-type population, whereas the PLP1-KO strain cannot be enriched, fitting the underlying phenotypes. The screen efficiency facilitates the isolation of new mutants from mutagenized parasite populations. The use of various egress enhancers will allow genetic dissection of the egress signaling pathways. This is illustrated by a mutant generated using dithitotreitol as an egress enhancer, which displays a defect in dithitotreitol induced egress but not in Ca(2+) ionophore induced egress.
Insights
Toxoplasma gondii egress involves complex signaling. A new screening method enriches for parasites with egress defects, enabling genetic dissection of these crucial signaling pathways.
Area of Science:
- Parasitology
- Cell Biology
- Molecular Biology
Background:
- Toxoplasma gondii egress from host cells is a critical lytic cycle event.
- This process is mediated by complex signaling in response to internal and external cues.
- The molecular mechanisms underlying T. gondii egress signaling remain largely uncharacterized.
Purpose of the Study:
- To develop and validate a screening procedure for identifying defects in Toxoplasma gondii induced egress.
- To facilitate the genetic analysis of signaling pathways governing parasite egress.
- To enable the isolation of novel mutants with impaired egress phenotypes.
Main Methods:
- Designed an in vitro screening assay for induced egress.
- Utilized glycans to prevent parasite reattachment and reinvasion.
- Employed PDTC (N,N'-[dithiobis(C2H4)]bis(phenylthiocarbamate)) to selectively eliminate extracellular parasites.
- Assessed screen efficiency using a temperature-sensitive mutant (F-P2) and a perforin-like protein 1 (PLP1) knockout mutant.
Main Results:
- The screening procedure achieved over 1000-fold enrichment for the F-P2 temperature-sensitive mutant.
- The PLP1-KO mutant showed no significant enrichment, consistent with its constitutive egress defect.
- A novel mutant with a defect in dithiotreitol-induced egress, but not Ca(2+) ionophore-induced egress, was isolated.
Conclusions:
- The developed screening method is effective for enriching T. gondii mutants with specific induced egress defects.
- This assay provides a powerful tool for the genetic dissection of T. gondii egress signaling pathways.
- Further application of this screen with various egress enhancers will uncover new components of the egress machinery.
