Генетические экрана изолировать Toxoplasma гондий Клетки-хозяина Мутанты Egress

Summary

Вперед генетики мощный метод, чтобы разгадать молекулярном уровне, как<em> Toxoplasma</em> Выходов из клетки-хозяина. Протоколы предоставляются химически mutagenize паразитов, обогащают для мутантов с дефектами в индуцированной выхода и проверки фенотип клонировали мутантов.

Abstract

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways^1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca²⁺, a signal that is also critical for host cell invasion^6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress⁹. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches^10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress¹³. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology^11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine^16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 °C), but fail to proliferate at the restrictive temperature (40 °C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype¹³. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites¹¹. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell¹⁹. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed²⁰. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

Protocol

Обзор Протоколы предоставляются сначала определить дозы мутагенов приводит к 70% гибели паразитов (протокол 1). Следующая процедура предусмотрена для обогащения индуцированных мутантов выход из мутагенизированный бассейн паразита (протокол 2, рисунок 2). Это сопровождает?…

Discussion

Описанный протокол обеспечивает эффективный способ изолировать Toxoplasma мутантов с выходным дефекта. Мы успешно изолировать мутантов по различным этапам пути выхода, некоторые из которых имеют двойное фенотип вторжения ^13. Потенциальное воздействие на вторжение можно опр?…

Materials

Name of the reagent	Company	Catalogue number	Comments
ENU	Sigma-Aldrich	N3385	1 M Stock in DMSO, store at -20°C
EMS	Sigma-Aldrich	M0880	1 M Stock in DMSO, store at -20°C
Dextran Sulfate	Sigma-Aldrich	D4911
PDTC	Sigma-Aldrich	P8765	100 mM Stock in PBS
Diff Quick	EMD Chemicals	65044-93
Filter holder	Cole-Parmer	540100
3 μm polycarbonate filter	Whatman Schleicher & Schuell	110612
Hemocytometer	Hausser Scientific	1475
CO2 incubators	Various manufacturers		Humidified, 5% CO2, at 35, 37 and 40°C
Fluorescence microscope	Various manufacturers		Ideally inverted, wide-field with 63x or 100x oil objective

HBSSc (according to Black et al.¹¹):

• 98.0 ml Hanks Balanced Salt Solution (Hyclone catalog number SH30588)
• 100 μl 1M MgCl₂ (100 mM end)
• 100 μl 1M CaCl₂ (100 mM end)
• 2.0 ml 1M Hepes pH 7.3 (20 mM end)
• 84 mg NaHCO₃ (10 mM end)