Department of Biology, Boston College
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Coleman, B. I., Gubbels, M. J. A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants. J. Vis. Exp. (60), e3807, doi:10.3791/3807 (2012).
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 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.
Protocols are provided to first define the dosage of the mutagen leading to a 70% killing of the parasites (protocol 1). The next procedure is provided to enrich the induced egress mutants from a mutagenized parasite pool (protocol 2, Figure 2). This is followed by a protocol to test the incidence of egress mutants in the enriched pool, or to validate the egress phenotype in individual mutants (protocol 3). Finally, a protocol is provided to generate single parasite clones from enriched populations by limiting dilution (protocol 4).
1. Titration of mutagen
Use special caution, such as double gloves, when working with highly mutagenic compounds and liquids. Collect liquid waste separately for proper disposal.
2. Enrichment of egress mutants (Figure 2)
3. Validation of mutant phenotypes by egress assays
After performing the enrichment screen and growing up the enriched population for one passage in HFF cells, the phenotypes need to be confirmed by an egress assay 11,13. This assay should also be used to validate single clones after protocol 4.
4. Clone egress mutants by limiting dilution
After validation of the phenotype of the enriched egress mutant population using protocol 3, the polyclonal population needs to be cloned to obtain single parasite clones.
5. Representative Results
The mutagens ENU and EMS are not stable when stored over long periods of time. Therefore, testing the mutagenic power of the stocks is critical to obtain reproducible results. Typical titration results and killing curves for both ENU and EMS are shown in Figure 1. However, it is recommended to perform plaque assays for every mutagenesis experiment to ascertain that the appropriate dosage was used. To maintain the diversity in the mutagenized parasite population, it is important to proceed with the egress mutant screen as quickly as possible. Typically, one passage of the parasites into a new flask is performed to let the surviving parasites recover before going forward with the screen. It should be kept in mind that doing this will result in division of the mutants. Therefore it cannot be excluded that multiple clonal mutants isolated after completing the whole procedure contain exactly the same genotype. To avoid isolating the same mutant multiple times it is recommended to isolate only a single egress mutant per mutagenesis, unless their phenotypes (e.g. differential egress inducer sensitivities) are very different from each other. On the other hand, not every screen results in isolation of mutants with the desired phenotype. In particular the Ca2+-ionophore A23187 resistant phenotype is rare and requires multiple mutagenesis experiments and screens to isolate a single egress mutant. Therefore it is recommended to perform 5-10 mutagenesis and screen experiments in parallel.
The enrichment power of the screen was tested by mixing a known egress mutant with wild-type parasites at different ratios. These mixes were subjected to the screen and the incidence of the mutant phenotype in the enriched population was assessed. By using wild-type parasites expressing cytoplasmic RFP and a mutant line expressing cytoplasmic YFP the incidences could quickly be established by flow cytometry (Figure 3)13. The results show that mutant phenotypes can be routinely enriched to 80% purity by starting with 1 egress mutant parasite per 10,000 wild-type parasites13. However, when the egress mutant: wild-type parasite start ratio is 1:100,000 parasites, the isolated population is only 1-2% (1:100) egress mutants. Therefore the enrichment power of the screen is 1,000-fold. Since the screen starts with inoculation of 120,000 parasites, of which typically 70% are viable, we typically perform two rounds of enrichment. The isolated parasites are grown up between the enrichment rounds. This procedure leads to a 100% mutant population even when starting with 1:1,000,000 egress mutant:wild-type parasites.
The egress assay to validate and characterize the phenotypes requires a multiplicity of host cell infection that allows differentiation of individual vacuoles. This is especially critical when analyzing conditions with high percentages of egress as the individual parasites are scattered around. As shown in Figure 4, if egressed populations are not well separated it is easy to underestimate the percentage of egress by counting two egressed vacuoles as one vacuole. Since egress and invasion are related processes, and some temperature-sensitive phenotypes display a mild phenotype at the lower temperature, not all mutants will have similar invasion efficiencies. Such mutants must therefore be inoculated at several fold higher dosages to obtain a high enough vacuole density for an accurate assessment of their egress phenotype. Furthermore, some egress inducers do not efficiently stimulate egress of vacuoles containing four parasites or less. As such, it is important that vacuoles contain eight parasites or more when starting the egress assay. In our lab we screen mutants isolated with a particular egress enhancer against other egress enhancers. By profiling their cross-reactivity the mutants can be grouped in different classes. Lastly, not all egress mutants will be temperature-sensitive. In particular mutants isolated with egress inducers triggering steps before the release of intracellular Ca2+ are prone to non-temperature sensitive phenotypes. This is due to the parallel pathways that can lead to egress before the signal converges on the release of intracellular Ca2+.
Figure 1. Chemical mutagen dosage titration. A. Plaque assays performed in a 6-well plate using various EMS concentrations and various numbers of parasites per well as indicated. The white spots are parasite plaques formed in the HFF monolayer. B, C. Survival curves of parasites upon exposure to various dosages of EMS (B) and ENU (C). Survival was assessed by plaque assays. A dosage inducing 70% killing is chosen for mutagenesis experiments. Averages of three independent experiments +/- standard deviation are shown.
Figure 2. Schematic representation of the egress mutant enrichment screen. Temperature is indicated on the y-axis, whereas time progression is represented by the x-axis. Green parasites reflect egress mutant parasites, red parasites reflect wild-type parasites. The timing of changes in conditions and stimulation by egress inducer are indicated at the top and are marked by arrows on the blue line reflecting the temperature profile. Adopted from Eidell et al.13.
Figure 3. Typical enrichment of egress mutant phenotypes. Enrichment results of an egress mutant mixed into wild-type parasites at various ratios (x-axis). The percentage of egress mutant phenotypes in the population grown up after the screen is plotted on the y-axis and was assessed by flow cytometry (egress mutant parasites expressed cytoplasmic YFP, wild-type parasites expressed cytoplasmic RFP). Results of three independent experiments are represented by red, blue and green data points. Adopted from Eidell et al.13.
Figure 4. Typical results of an egress assay. A. Arrows mark four different intact vacuoles containing 4-8 parasites. B. Arrows mark four groups of scattered parasites reflecting four independent egressed vacuoles. Egress was induced by A23187 (B) or DMSO as a negative control (A). Parasites express cytoplasmic YFP22.
Figure 5. Serial dilution for clonal parasite lines. Four polyclonal populations (red, blue, yellow, green) are serially diluted in a single 384-well plate confluent with HFF cells. Row C and I receive 10 parasites per well and are 2-fold diluted (40 μl + 40 μl) until rows H and N, respectively. The shade of grey indicates the decreasing number of parasites per well. Typically, single clones are found in rows D-F and J-L.
|Compound||Stock||Working concentration||μl needed for T25 (10 ml)||Incubation time (min)|
|DTT||1 M in DMSO||5 mM||50||15|
|A23187||2 mM in DMSO||1 μM||5||5|
|nigericin*||2 mM in DMSO||10 μM||50||30|
*nigericin cannot be used in the enrichment screen as the parasites do not survive nigericin stimulation; only use nigericin in the validation of egress mutants.
Table 1. Egress inducers used in procedures. Dilute in 10 ml HBBSc containing 25 mg/ml DS for the screen, or no DS for mutant validation (egress assay). All compounds from Sigma-Aldrich.
The described protocol provides an efficient method to isolate Toxoplasma mutants with an egress defect. We have successfully isolated mutants along various steps of the egress pathway, some of which have a dual invasion phenotype13. Potential effects on invasion can be determined using the so-called red-green assay, which differentiates invaded from non-invaded parasites by differential antibody staining23,24. For both invasion and egress assays it could be convenient to express an autofluorescent protein marker in the cytoplasm of the parasites13,22. However, if the parasites being mutagenized already express a fluorescent protein this could interfere with additional phenotypic characterization by immunofluorescence later on. It is always possible to add a fluorescent marker to a mutant after its isolation. As described, a simple, non-fluorescent alternative is Diff-Quick staining for egress.
Historically, the mutagen ENU has been applied to Toxoplasma genetics14. However, ENU preferentially targets AT base pairs25, which we validated in Toxoplasma (Farrell, Marth, Gubbels et al., manuscript submitted). Since the frequency of A/T is lower in amino acid coding sequence than non-coding sequence, the majority of mutations induced by ENU will be in non-coding sequence. However, EMS typically has a preference for GC base pairs, i.e. coding sequences. The vast majority of temperature-sensitive mutations originate in codon-changing mutations, which is a reason to use EMS over ENU.
Although a variety of egress inducers triggering different aspects of the signaling pathway are described in Table 1, there are other pharmacological agents known to act on processes required for egress. For instance, caffeine has been shown to induce microneme secretion in extracellular parasites, which is required for both egress and invasion7,26,27. However, caffeine cannot be used to trigger egress from infected host cells, likely because caffeine does not efficiently diffuse through the host cell toward the parasites residing in a vacuolar compartment. Similarly, one egress trigger is the accumulation of abscisic acid, but it is impossible to screen for mutants in this pathway since abscisic acid does not diffuse into the host cell28. And as indicated in Table 1, the K+-ionophore nigericin is an efficient egress inducer5, however, parasites do not survive nigericin stimulation. Therefore this compound is also not suitable for the screen. In an alternative, more elaborate screening procedure for ionophore mutants it was reported that many mutants with a delay in ionophore induced egress survive prolonged extracellular exposure to ionophore11. However, no egress mutants resistant to ionophore induced egress were isolated in this screen, making the phenotypes weaker. It is at present unclear whether mutants with resistance to prolonged exposure to other egress inducers can be obtained.
Since there appears to be several parallel pathways that can lead to egress, the question arises as to which one would be the most relevant in an (experimental) infection. It appears that immune system attacks on the infected cell are the main trigger of egress29. It is possible to use such triggers in the mutant screen. For instance, CD8 T-cells can stimulate egress through both a Fas-mediated and a perforin-mediated pathway30. Therefore, by using a FasL expressing human T-cells as a host cell in the egress screen, egress can be triggered by incubation with an anti-Fas antibody to enrich for mutants in this pathway.
To genetically map the mutations underlying the egress phenotypes, two different approaches are available in Toxoplasma. Unlike genetic model organisms, allelic segregation by crossing is not an option since the sexual cycle of the parasite can only be triggered in the gut of the cat. Aside from the impracticality and poor efficiency of this process, parasite strains grown in the lab very quickly lose the ability to infect cats. To bypass this problem an efficient wild-type cosmid library complementation approach has been developed, which complemented 90% of cell division mutants18. In addition, we recently established complete genome resequencing protocols to identify all mutations induced in the genome (Farrell, Marth, Gubbels et al., manuscript submitted). Typically we identify 20-70 single nucleotide polymorphisms in chemically induced mutants. Between these two approaches we have been able to identify the causative mutation in all chemically induced mutants characterized to date.
Taken together, the described screen provides a versatile tool that will allow forward genetic dissection of the egress pathways. We expect this will identify several components of the signaling pathway known to exist based on pharmacological data, but for which no good candidate genes could be identified in the Toxoplasma genome.
We have nothing to disclose.
This work was funded by American Heart Association Scientist Development Grant 0635480N and National Institutes of Health research grant AI081220. BIC is supported by a Knights Templar Eye Foundation research grant.
|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|
|PDTC||Sigma-Aldrich||P8765||100 mM Stock in PBS|
|Diff Quick||EMD Chemicals||65044-93|
|3 μm polycarbonate filter||Whatman Schleicher & Schuell||110612|
|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):