A Genetic Screen to Isolate <em>Toxoplasma gondii</em> Host-cell Egress Mutants

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 pathways1-5. 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 invasion6-8. 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 approaches10-12. 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 biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. 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-reinvasion 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.

Guanabenz Repurposed as an Antiparasitic with Activity against Acute and Latent Toxoplasmosis

“…At 4 h postinfection, extracellular parasites were removed and the medium was replaced with medium containing the indicated concentration of drug or vehicle. At 32 h postinfection, parasites were fixed with 1 ml ice-cold methanol for 10 min and then stained with Diff-Quick (Siemens) for 1 min to visualize parasites by light microscopy (17). The number of tachyzoites in each of 50 randomly chosen vacuoles was recorded.…”

Forward Genetics Screens Using Macrophages to Identify <em>Toxoplasma gondii </em>Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

“…NOTE: Detailed protocols for chemical mutagenesis 38 , isolation of parasites by limiting dilution 38 , isolation of murine bone marrow derived macrophages 39 , growth of T. gondii in human foreskin fibroblast (HFF) cells and cyst production in macrophages and basic immunofluorescence analysis (IFA) 32 are referenced. Carry out all cell culture at 37 °C in 5% CO 2 in D10 media (Dulbecco’s Modified High Glucose Eagle Medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin).…”

“…Insertion of a plasmid into a gene is also likely to disrupt the function of a gene in contrast to chemical mutagenesis that typically results in single nucleotide changes. However, chemical mutagenesis with either N-ethyl-N-nitrosourea (ENU) or ethylmethane sulfonate (EMS) may offer an increased ability to analyze a larger portion of the parasite genome, compared to insertional mutagenesis, as it creates multiple single nucleotide polymorphisms (estimated at 10 -100) per mutant 34,38 . Moreover, recent advances in whole genome profiling has made it possible to use next generation sequencing to identify the most likely candidate genes responsible for the identified phenotype of a mutated parasite 34,38 .…”

“…However, chemical mutagenesis with either N-ethyl-N-nitrosourea (ENU) or ethylmethane sulfonate (EMS) may offer an increased ability to analyze a larger portion of the parasite genome, compared to insertional mutagenesis, as it creates multiple single nucleotide polymorphisms (estimated at 10 -100) per mutant 34,38 . Moreover, recent advances in whole genome profiling has made it possible to use next generation sequencing to identify the most likely candidate genes responsible for the identified phenotype of a mutated parasite 34,38 . Regardless of the mutagenesis approach, confirmation of the role of the parasite gene in resistance to macrophage activation ultimately requires gene deletion and complementation to fulfill molecular Koch’s postulates.…”

Genetic Evidence for Cytochrome b Q i Site Inhibition by 4(1 H )-Quinolone-3-Diarylethers and Antimycin in Toxoplasma gondii

“…N-Nitroso-N-ethylurea (ENU) chemical mutagenesis and ELQ-316 selective pressure were applied to the RH Δuprt strain of T. gondii to select for ELQ-resistant clones as previously described (15,23). The RH Δuprt strain was used after several attempts to select ELQ-316-resistant clones of RH strain T. gondii resulted in the isolation of clones without sustained resistance.…”