Development of a screen to dissect Toxoplasma gondii egress

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 Ca2+ ionophore induced egress. (C) 2010 Elsevier B.V. All rights reserved.