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.

Overview
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.
<ol>
 <li>Inoculate T25 tissue culture flasks confluent with human foreskin fibroblast (HFF) cells with 1 ml of freshly lysed tachyzoites and grow 18-25 hr at 37 &deg;C under 5% CO2 in Ed1 medium (D-MEM supplemented with 1% heat-inactivated fetal bovine serum, 0.2 mM L-glutamine, 50 U/ml penicillin, 50 &mu;g/ml streptomycin, and 0.25 &mu;g/ml Amphotericin B). See Roos et al. for general growth and media of HFF cells and parasites21.</li>
 <li>Replace medium with 10 ml 0.1% Fetal Bovine Serum medium (dilute Ed1 1:10 in D-MEM) and leave in humidified 37 &deg;C incubator under 5% CO2 (called "37 &deg;C incubator" from here on) for 10 min.</li>
 <li>Add 0, 12.5, 25, 50, or 100 &mu;l ENU (1 M Stock in DMSO) or EMS (1 M Stock in DMSO) per flask (mutagen working dilutions will be 1.25, 2.5, 5, and 10 mM, respectively). Add DMSO to 100 &mu;l for each flask. Incubate for 4 hrs in a 37 &deg;C incubator.</li>
 <li>Wash three times for 10 seconds at room temperature by rinsing the monolayer with 10 ml cold PBS (pre-cooled at 4 &deg;C).</li>
 <li>Add 5 ml PBS, scrape the monolayer loose with a rubber policeman (cell scraper), pass the scraped cells through a 26.5 G needle to physically remove the parasite from the fibroblasts (clip off the shaft outside the needle with heavy duty scissors to not expose the needle), and filter with 3.0 &mu;m polycarbonate filter. Multiple needle passages will increase the efficiency of releasing parasites from the host cell.</li>
 <li>Count the parasite concentration using a hemocytometer. Let the parasites settle for 5 min in the hemocytomer before counting.</li>
 <li>Dilute parasites to 10,000 parasites per 3 ml in Ed1 (for 10 ml 33,333 parasites are needed).</li>
 <li>Inoculate one well in a 6 well plate with 3 ml of the diluted parasites (containing 10,000 parasites). Serially dilute the parasites 10-fold over 3 wells in a 6-well plate confluent with HFF cells containing 2.7 ml Ed1 by transferring 300 &mu;l out of the first well. Leave plates undisturbed in the 37 &deg;C incubator for 7 days.</li>
 <li>Aspirate medium, fix 15 min with 3 ml/well 100% ethanol, stain with 3 ml/well crystal violet solution (12.5 g crystal violet in 125 ml ethanol mixed with 500 ml 1% ammonium oxalate) for 15 min, rinse with 3 ml/well PBS (1 minute) and air dry. All at room temperature.</li>
 <li>Count plaques and select concentration of mutagen needed to achieve survival of 30% of the exposed parasites (70% killing dosage: see Figure 1). A dosage of 70% killing has been used historically14 and induced less than 100 point mutations per genome (Farrell, Marth, Gubbels et al., manuscript submitted).</li>
</ol>
2. Enrichment of egress mutants (Figure 2)
<ol>
 <li>Perform mutagenesis as described above using a mutagen dosage inducing 70% killing. Grow up the mutagenized population for one passage in a new flask of host cells.</li>
 <li>Infect a T25, HFF confluent tissue culture flask with 120,000 freshly lysed parasites from a mutagenized population in 10 ml Ed1. Incubate for 2 hours in a 35 &deg;C incubator under 5% CO2 and humidified (from hereon called "35 &deg;C incubator").</li>
 <li>Aspirate medium and rinse 10 seconds with 10 ml cold PBS and then add 10 ml Ed1 medium supplemented with 25 mg/ml dextran sulfate (DS)13. Incubate for 26 hours in a 40 &deg;C incubator under 5% CO2 and humidified (from hereon called "40 &deg;C incubator").</li>
 <li>Prepare working solution of egress enhancers. Dilute the egress inducer of choice at the working concentration in a 15 ml Falcon tube containing 10 ml HBSSc supplemented with 25 mg/ml DS. Pre-warm the dilutions for 30 min in a 37 &deg;C waterbath. See Table 1 for working concentrations.</li>
 <li>Aspirate medium from the parasite-infected flasks and add pre-warmed egress inducer solution. Incubate in the 37 &deg;C incubator for the times indicated in Table 1.</li>
 <li>Aspirate medium and rinse 10 seconds with 10 ml cold PBS and then add 10 ml Ed1 supplemented with 25 mg/ml DS and 50 &mu;M pyrrolidine dithiocarbamate (PDTC: add 5 &mu;l of 100 mM PDTC Stock in PBS)13. Incubate 5 hours in a 35 &deg;C incubator.</li>
 <li>Aspirate medium and rinse 10 seconds once with 10 ml PBS (room temperature) and then add 10 ml Ed1 medium. Put flasks back into the 35 &deg;C incubator until parasites destroy the monolayer: shake flasks daily. This recovery takes around 7 days.</li>
</ol>
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.
<ol>
 <li>Inoculate 20,000 parasites per well into a 24-well plate containing confluent HFF cells grown on coverslips (1 ml Ed1 medium per well). Incubate 8 hrs in a 35 &deg;C incubator then transfer into a 40 &deg;C incubator for 24 hrs.</li>
 <li>Wash with 1 ml/well PBS (10 seconds at room temperature). Add 1 ml egress inducers (include a DMSO only negative control), pre-warmed and diluted in HBSSc and incubate for the times described in Table 1.</li>
 <li>Aspirate medium and fix with 1 ml/well 100% methanol for 15 min at room temperature.</li>
 <li>If parent parasites expressing an autofluorescent protein were used for the mutagenesis, proceed to step #3.513. If non-fluorescent protein expressing parasites were used as parent line, stain the fixed coverslips with Diff-Quick stain for 1 min at room temperature11.</li>
 <li>Wash 5 min with 1 ml/well PBS in the 24-well plate at room temperature.</li>
 <li>For fluorescent protein expressing parasites: quickly rinse the coverslip in ddH2O (dipping) and mount on slides under gelmount to protect the fluorescence signal. For Diff-Quick stained parasites, wash 10 seconds in 100% ethanol and let air-dry before mounting on slide.</li>
 <li>Using a (fluorescence) microscope with a 40-60x objective count the percentage of vacuoles egressed versus the vacuoles that stayed intracellular (see Figure 4).</li>
</ol>
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.
<ol>
 <li>Count parasite population in a hemocytometer and dilute to a concentration of 500 parasites per ml in Ed1 medium.</li>
 <li>In a 384-well plate containing a confluent monolayer of HFF cells, replace the medium with 40 &mu;l/well of Ed1 medium using a multi-channel pipet. As shown in Figure 5, four polyclonal populations can be cloned per plate as follows: pipet 40 &mu;l/well of diluted mutant 1 into wells C3-C12, mutant 2 into wells C13-C22, mutant 3 into wells H3-H12, and mutant 4 into wells H13-H22 (end volume in wells is now 80 &mu;l). Using a multi-channel pipet, pipet the solution up and down 5 times, then transfer 40 &mu;l to the row below the starting row (from row C to D or row H to I). Continue these 2-fold serial dilutions through row G (mutants 1 and 2) or row N (mutants 3 and 4). Discard the extra 40 &mu;l from the last row. Incubate in a 35 &deg;C incubator for 7-10 days without disturbing the plate.</li>
 <li>Check the wells on an inverted microscope with a 10-20x objective for the presence of single plaques, visible as 'holes' in the monolayer.</li>
 <li>Pick 4 wells per mutant with a single plaque and transfer the parasites into a tissue culture flask confluent with HFF cells and filled with Ed1 medium. Grow the parasites up in a 35 &deg;C incubator; shake the flasks daily to disperse the extracellular parasites. This typically takes 7 days.</li>
</ol>
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+.
<img alt="Figure 1" src="/files/ftp_upload/3807/3807fig1.jpg" /> 
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.
<img alt="Figure 2" src="/files/ftp_upload/3807/3807fig2.jpg" /> 
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.
<img alt="Figure 3" src="/files/ftp_upload/3807/3807fig3.jpg" /> 
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.
<img alt="Figure 4" src="/files/ftp_upload/3807/3807fig4.jpg" /> 
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.
<img alt="Figure 5" src="/files/ftp_upload/3807/3807fig5.jpg" /> 
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 &mu;l + 40 &mu;l) 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.
<table border="1">
 <tbody>
 <tr>
 <td>Compound</td>
 <td>Stock</td>
 <td>Working concentration</td>
 <td>&mu;l needed for T25 (10 ml)</td>
 <td>Incubation time (min)</td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>1 M in DMSO</td>
 <td>5 mM</td>
 <td>50</td>
 <td>15</td>
 </tr>
 <tr>
 <td>ethanol</td>
 <td>190 Proof</td>
 <td>5%</td>
 <td>500</td>
 <td>30</td>
 </tr>
 <tr>
 <td>A23187</td>
 <td>2 mM in DMSO</td>
 <td>1 &mu;M</td>
 <td>5</td>
 <td>5</td>
 </tr>
 <tr>
 <td>nigericin*</td>
 <td>2 mM in DMSO</td>
 <td>10 &mu;M</td>
 <td>50</td>
 <td>30</td>
 </tr>
 </tbody>
</table>
 
*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.

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+membrane+disruption+by+a+perforin-like+protein+facilitates+parasite+exit+from+host+cells.">Rapid membrane disruption by a perforin-like protein facilitates parasite exit from host cells.</a> Science. 323 (5913), 530-533 (2009).</li><li>Lovett, J. L., Marchesini, N., Moreno, S. N., Sibley, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+gondii+microneme+secretion+involves+intracellular+Ca(2+)+release+from+inositol+1,4,5-triphosphate+(IP(3))/ryanodine-sensitive+stores.">Toxoplasma gondii microneme secretion involves intracellular Ca(2+) release from inositol 1,4,5-triphosphate (IP(3))/ryanodine-sensitive stores.</a> J. Biol. Chem. 277 (29), 25870-25876 (2002).</li><li>Nagamune, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abscisic+acid+controls+calcium-dependent+egress+and+development+in+Toxoplasma+gondii.">Abscisic acid controls calcium-dependent egress and development in Toxoplasma gondii.</a> Nature. 451 (7175), 207-210 (2008).</li><li>Tomita, T., Yamada, T., Weiss, L. M., Orlofsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Externally+triggered+egress+is+the+major+fate+of+Toxoplasma+gondii+during+acute+infection.">Externally triggered egress is the major fate of Toxoplasma gondii during acute infection.</a> J. Immunol. 183 (10), 6667-6680 (2009).</li><li>Persson, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Death+receptor+ligation+or+exposure+to+perforin+trigger+rapid+egress+of+the+intracellular+parasite+Toxoplasma+gondii.">Death receptor ligation or exposure to perforin trigger rapid egress of the intracellular parasite Toxoplasma gondii.</a> J. Immunol. 179 (12), 8357-8365 (2007).</li></ol>

We have nothing to disclose.

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 autoflu...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>ENU</td>
 <td>Sigma-Aldrich</td>
 <td>N3385</td>
 <td>1 M Stock in DMSO, store at -20&deg;C</td>
 </tr>
 <tr>
 <td>EMS</td>
 <td>Sigma-Aldrich</td>
 <td>M0880</td>
 <td>1 M Stock in DMSO, store at -20&deg;C</td>
 </tr>
 <tr>
 <td>Dextran Sulfate</td>
 <td>Sigma-Aldrich</td>
 <td>D4911</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>PDTC</td>
 <td>Sigma-Aldrich</td>
 <td>P8765</td>
 <td>100 mM Stock in PBS</td>
 </tr>
 <tr>
 <td>Diff Quick</td>
 <td>EMD Chemicals</td>
 <td> 65044-93</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Filter holder</td>
 <td>Cole-Parmer</td>
 <td>540100</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>3 &mu;m polycarbonate 			filter</td>
 <td>Whatman
 Schleicher &amp; Schuell</td>
 <td>110612</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Hemocytometer</td>
 <td>Hausser Scientific</td>
 <td>1475</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>CO2 incubators</td>
 <td>Various manufacturers</td>
 <td>&nbsp; </td>
 <td>Humidified, 5% CO2, at 35, 37 and 40&deg;C</td>
 </tr>
 <tr>
 <td>Fluorescence microscope</td>
 <td>Various manufacturers</td>
 <td>&nbsp; </td>
 <td>Ideally inverted, wide-field with 63x or 100x oil 			objective</td>
 </tr>
 </tbody>
</table>
HBSSc (according to Black et al.11):
<ul>
<li>98.0 ml Hanks Balanced Salt Solution (Hyclone catalog number SH30588)</li>
<li>100 &mu;l 1M MgCl2 (100 mM end)</li>
<li>100 &mu;l 1M CaCl2 (100 mM end)</li>
<li>2.0 ml 1M Hepes pH 7.3 (20 mM end)</li>
<li>84 mg NaHCO3 (10 mM end) </li>
</ul>

a genetic screen to isolate toxoplasma gondii host-cell egress mutants

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 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;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.

Watch this Scientific Journal Video about A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants at JoVE.com

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

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Immunology and Infection

12.9K Views.  Boston College. Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

Video: A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with ...

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

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

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

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

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...