The authors would like to thank Drs. Sibley and Carruthers for sharing pSAG1-Cas9-sgRNA-TgUPRT plasmid and anti-TgCPL and TgActin antibodies. This work was supported by the Clemson Startup fund (to Z.D.), Knights Templar Eye Foundation Pediatric Ophthalmology Career-Starter Research Grant (to Z.D.), a pilot grant of an NIH COBRE grant P20GM109094 (to Z.D.), and NIH R01AI143707 (to Z.D.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Toxoplasma gondii is categorized in Risk Group 2 and must be handled at a Biosafety Level 2 (BSL-2). The protocol has been reviewed and approved by the Institutional Biosafety Committee at Clemson University.
1. Luciferase-based Toxoplasma growth assay
<ol>
	<li>Seed human foreskin fibroblasts (HFFs) 1 week before parasite inoculation to ensure that host cells are fully confluent. Perform a mock assay in a transparent plate to ensure that parasites remain intracellular throughout the evaluation period. 
	NOTE: Here, the assay is conducted in 96 well microplates. According to experimental needs, it can be scaled up to 384 or 1536 well microplates.</li>
	<li>Pass Toxoplasma parasites into confluent HFFs 2 days prior to use by transferring ~0.3-0.4 mL of fully lysed parasites into a T25 flask. Incubate infected host cells at 37 &#176;C with 5% CO2 for 2 days.</li>
	<li>Syringe 5 mL of freshly lysed parasites through a 21 G safety needle 5x to liberate intracellular parasites, then pass through a 3 &#181;m filter to remove host cell debris. Rinse residual parasites out of the flask using 7 mL of phenol red-free D10 medium, then pass through the filter again.</li>
	<li>Centrifuge parasites at 1000 x g for 10 min at room temperature (RT). Pour off the supernatant and resuspend the pellet in 10 mL of phenol red-free D10 media.</li>
	<li>Count parasites using a hemocytometer to determine the concentration.</li>
	<li>Dilute parasites to 1 x 104 parasites/mL for the wild-type (WT) strain. For growth-deficient parasite strains, increase the concentration accordingly to observe a significant increase in luciferase signals.</li>
	<li>Aspirate media carefully from 96 well microplates pre-seeded with HFFs and inoculate 150 &#181;L of parasite resuspension into wells in a format of three columns and five rows, which represents three technical replicates and five timepoints.</li>
	<li>Incubate the microplate at 37 &#176;C and 5% CO2 for 4 h.</li>
	<li>Aspirate media carefully from the wells to remove non-invaded parasites, then fill the wells with RT phenol red-free media in each row (except for the first row).</li>
	<li>Mix equal volumes of PBS and 2x luciferase assay buffer and dilute the luciferase substrate to 12.5 &#181;M.</li>
	<li>Add 100 &#181;L of dilute luciferase substrate into each well of the top row. Incubate the microplates at RT for 10 min to allow the cells to fully lyse.</li>
	<li>Measure the luciferase activity using a microplate reader. The plate reader settings are listed in Table 1. Each reading represents the initial number of invaded parasites at 4 h post-infection.</li>
	<li>Repeat steps 1.9-1.12 for each row every 24 h for 4 days without changing the medium. These readings reflect the total number of replicated parasites at 24 h, 48 h, 72 h, and 96 h post-infection.</li>
	<li>Calculate the average readings at each timepoint and divide them by the average readings at 4 h to determine the fold changes in parasite growth over time.</li>
	<li>Plot the data using graphing software. A representative growth reading table and plots of RH&#916;ku80::NLuc parasites are shown in Figure 1A,B.</li>
	<li>To calculate doubling time, plot the log2 values of fold changes at the individual timepoints over the incubation time. Use a linear regression function to calculate slope, which represents the doubling time of each strain (Figure 1A,C).</li>
</ol>
2. Evaluation of chemical compound inhibition efficacy against Toxoplasma growth
NOTE: Here, evaluation of the inhibition of LHVS in Toxoplasma growth is presented as an example. Eight different concentrations of LHVS are tested, and three technical replicates are performed for each of the three biological replicates for both RH&#916;ku80::NLuc and RH&#916;ku80&#916;cpl::NLuc strains.
<ol>
	<li>Prior to the parasite infection, seed HFFs to 96 well microplates in the format of three rows and nine columns for one biological replicate per compound per strain. Host cells will be allowed to grow for at least 7 days before use.</li>
	<li>Pass RH&#916;ku80::NLuc and RH&#916;ku80&#916;cpl::NLuc parasites for 2 days prior to use. Follow steps 1.2-1.6 for parasite purification and quantification. Resuspend parasites in phenol red-free media at 1 x 104 parasites/mL.</li>
	<li>Aspirate media from a plate containing&#160;confluent HFFs and inoculate each well with 150 &#181;L of parasite resuspension. Incubate the microplate at 37 &#176;C and 5% CO2 for 4 h.</li>
	<li>Prepare LHVS at eight different concentrations in a 12 well reservoir by serial dilution. Generally, the concentrations are decreased by three-fold in a serial dilution manner. 
	NOTE: The lowest concentration is reduced by 6,561-fold relative to the highest concentration. The fold change of the dilution can be adjusted accordingly based on different properties of individual compounds.</li>
	<li>At 4 h post-infection, aspirate media to remove non-invaded parasites and fill each well from columns 2-9 with 150 &#181;L of media supplemented with LHVS at different concentrations. Leave the first column filled with regular medium to serve as a nontreated control.</li>
	<li>Incubate the microplate at 37 &#176;C and 5% CO2 for an additional 96 h.</li>
	<li>Perform steps 1.9-1.11 and measure luciferase activity of individual wells.</li>
	<li>Average the luciferase activities of three technical replicates from wells of each individual LHVS concentration.</li>
	<li>Divide the average luciferase activity for each LHVS concentration by the average luciferase activity derived from nontreated parasites to calculate the normalized luciferase activity as a percentage.</li>
	<li>Plot the normalized luciferase activities against the individual LHVS concentrations using graphing software (Figure 2). Inhibition of pyrimethamine against parasite growth is also measured as a control. Pyrimethamine is a clinical antibiotic used to treat acute toxoplasmosis by inhibiting folic acid metabolism in Toxoplasma.</li>
	<li>Calculate the IC50 values for individual compounds using the embedded method in the graphing software, normalized response vs. [inhibitor], under the &#34;dose-response-inhibition&#34; regression program. The IC50 is calculated using the following formula: 
	Y = 100/(1 + X/IC50) 
	Where: Y represents normalized luciferase activities of infected cells under different concentrations of inhibitor, and X represents individual concentrations of inhibitor.</li>
</ol>
3. CRISPR-Cas9-based gene deletion in Toxoplasma parasites
<ol>
	<li>Generation of a plasmid construct expressing guide RNA (sgRNA) and Cas9 for deleting a gene of interest
	<ol>
		<li>Go to <a href="http://www.ToxoDB.org/" target="_blank">www.ToxoDB.org</a> and retrieve the entire gene coding sequence, including introns and exons, along with 1.5 kb 5&#39;-UTRs and 3&#39;-UTRs (untranslated regions). 
		NOTE: Here, TgCPL (TGGT1_321530) is targeted as a representative example.</li>
		<li>Copy the retrieved TgCPL sequence into the sequence analysis software (refer to Table of Materials for the name and version) and label the 5&#39;- and 3&#39;-UTR regions.</li>
		<li>Select the Tools icon in the top menu bar, then select Cloning | Find CRISPR Sites.</li>
		<li>Choose 3&#39;(Cas9)&#39; for the PAM site location and select the folder containing the Toxoplasma genome sequence in the specificity scoring section. Leave the rest of the settings as defaults.</li>
		<li>Choose a sgRNA with the following two criteria: 1) showing a high specificity score, generally &#62;98%, and 2) lacking a G following the NGG, a protospacer adjacent motif (PAM) sequence. The selected sgRNA is usually located at sites close to the start and stop codons of the gene of interest.</li>
		<li>Copy the sequence of the selected sgRNA and paste it into the following primer template. 
		<img src="/files/ftp_upload/60985/60985eq1.jpg" /> 
		The portion in red represents the selected TgCPL sgRNA sequence. It can be replaced with different sgRNAs for various genes of interest. 
		NOTE: If the selected sgRNA does not start with G, add G at the beginning of the sgRNA to help enhance its expression.</li>
		<li>Perform a PCR reaction to modify the pre-existing plasmid expressing sgRNA (Figure 3A) that targets Toxoplasma uracil phosphoribosyltransferase (TgUPRT) gene23 using a PCR premix with the settings provided in Table 2.</li>
		<li>Run the PCR product on an agarose gel to confirm successful amplification. A 10 kb PCR product is expected to be amplified (Figure 3B).</li>
		<li>Extract the PCR product using a DNA gel extraction kit and circularize it using a site-directed mutagenesis kit. Refer to Table 3 for the recipe. Incubate the reaction for 10-20 min at RT.</li>
		<li>Transform the circularized PCR product into E. coli and pick 10 clones for further verification of incorporation of designed sgRNA.</li>
		<li>Grow two clones and extract plasmids. Cut the purified plasmids with BamHI and EcoRV. The candidate plasmids will yield two bands at 2.4 kb and 7.2 kb (Figure 3C).</li>
		<li>Send the plasmids for Sanger sequencing using M13 reverse primers to confirm successful replacement of TgUPRT sgRNA with the designed sgRNA (Figure 3D).</li>
	</ol>
	</li>
	<li>Generation of repair template for gene deletion via HDR mechanism
	<ol>
		<li>According to the targeting sites of the selected sgRNA, locate 50 bp of 5&#39;-UTRs or 3&#39;-UTRs of the target gene for homology-dependent recombination (HDR, see discussion section). The selection of regions follows the criteria listed below, depending on the location&#160;the sgRNA targets.
		<ol>
			<li>If the cleavage site by Cas9 is located upstream from the start codon, select the following: a 50 bp DNA sequence upstream from the cleavage site as the left HDR region, and a 50 bp DNA sequence downstream from the stop codon as the right HDR region.</li>
			<li>If the cleavage site by Cas9 is between the start and stop codons, select the following: a 50 bp DNA sequence upstream from the start codon as the left HDR region, and a 50 bp DNA sequence downstream from the stop codon as the right HDR region.</li>
			<li>If the cleavage site by Cas9 is located downstream from the stop codon, select the following: a 50 bp DNA sequence upstream from the start codon as the left HDR region, and a 50 bp DNA sequence downstream from the cleavage site as the right HDR region. 
			NOTE: For the TgCPL gene, the cleavage site is located between the start and stop codons. Thus, the following primers are designed for amplifying the repair template using pMDC64 as the template, which encodes a pyrimethamine resistance cassette. The sequences in black anneal to the pMDC64 plasmid for PCR amplification. The regions labeled in red are TgCPL-specific sequences for homologous recombination. 
			<img src="/files/ftp_upload/60985/60985eq2.jpg" /></li>
		</ol>
		</li>
		<li>Perform PCR using a PCR premix under the PCR conditions described in Table 4.</li>
		<li>Run the PCR product on an agarose gel (Figure 3E), followed by gel extraction and standard nucleic acid quantification procedures. 
		NOTE: If the expected band cannot be successfully amplified, optimize PCR conditions and/or switch primer pairs.</li>
	</ol>
	</li>
	<li>Toxoplasma transfection
	<ol>
		<li>Pass RH&#916;ku80::NLuc parasites for 2 days in a T25 flask containing confluent HFFs. A T25 flask of fully lysed parasites is sufficient for two to three transfections.</li>
		<li>Syringe and filter-purify parasites as described in step 1.2. Resuspend parasites in cytomix buffer and spin down at 1,000 x g for 10 min at RT.</li>
		<li>Wash pelleted parasites with 10 mL of cytomix&#160;buffer and spin down the parasites at 1,000 x g for 10 min at RT.</li>
		<li>Carefully pour off the supernatant and resuspend the parasites in the same buffer at a concentration of 1 x 108 parasites/mL.</li>
		<li>Mix 2 &#181;g of repair template DNA with 20 &#181;g of the sgRNA/Cas9 expression plasmids (mass ratio = 1:5, equivalent to a 1:3 molar ratio). If the amplification yield of repair template is low, reduce the input of both DNA pieces accordingly. A minimum of 0.5 &#181;g of repair template can be used.</li>
		<li>Mix 400 &#181;L of parasite resuspension, DNA, and 5 &#181;L of 200 mM ATP/500 mM reduced glutathione (GSH) in a 1.5 mL centrifuge tube. Bring the total volume to 500 &#181;L with cytomix buffer, if needed.</li>
		<li>Transfer the mixture of parasites and DNA to an electroporation cuvette (4 mm gap width) and perform electroporation (2 kV voltage, 50&#160;&#937; resistance) using an electroporation apparatus.</li>
		<li>Transfer electroporated parasites to a T25 flask containing confluent HFFs in fresh D10 medium. Apply appropriate antibiotic for drug selection after 24 h.</li>
		<li>Keep&#160;drug selective pressure until the growth of the transgenic parasites is stable.</li>
		<li>Purify genomic DNA from the knockout population and check for integration of the pyrimethamine resistance cassette into the TgCPL locus by PCR. After&#160;verified, proceed to section 3.4. If not, perform another round of parasite transfection and drug selection. Inability to detect the correct integration of the drug resistance cassette usually suggests that the target gene is essential or that the gene locus is not accessible.</li>
	</ol>
	</li>
	<li>Cloning of knockout parasites
	<ol>
		<li>Seed two 96 well microplates with HFF cells and incubate at 37 &#176;C and 5% CO2 for 1 week prior to cloning parasites.</li>
		<li>Pass ~0.3-0.4 mL of the population of transgenic parasites in a T25 flask containing confluent HFFs and grow them for 2 days. Consider passing more parasites if the mutant shows growth defects. 
		NOTE: To achieve the best yield and viability, the host cells are heavily infected by the parasites, and most of the parasites are kept in the intracellular stage.</li>
		<li>Syringe infected host cells and filter-purify freshly lysed parasites as mentioned in step 1.3. Resuspend the parasites in D10 medium and spin them down at 1,000 x g for 10 min at RT.</li>
		<li>Resuspend the pelleted parasites in 10 mL of D10 medium.</li>
		<li>Count parasites using a hemocytometer to determine the parasite concentration.</li>
		<li>Conduct a two-step dilution to bring the concentration to 10 parasites/mL in D10 medium supplemented with the appropriate antibiotic. Usually, the initial parasite resuspension is diluted by 1,000-fold, followed by a second dilution to 10 parasites/mL.</li>
		<li>Aspirate media from 96 well microplates containing confluent HFFs and inoculate 150 &#181;L of diluted parasites into each well.</li>
		<li>Incubate plates at 37 &#176;C with 5% CO2 for 7 days without disturbance to allow plaque formation. The incubation period can be longer if transgenic parasites exhibit growth defects.</li>
		<li>Screen the plates using a phase-contrast microscope and mark only the wells containing a single plaque.</li>
		<li>Perform colony PCR to identify correct clones.
		<ol>
			<li>Use pipette tips to scrape the bottom of each well to lift infected HFF monolayers.</li>
			<li>Pipet 75 &#181;L of the cell resuspension from each marked well into 1.5 mL microcentrifuge tubes.</li>
			<li>Centrifuge tubes for 10 min at maximum speed at RT. Carefully aspirate the supernatant and resuspend the pellet in 10.25 &#181;L of lysis buffer containing dilution buffer and DNA release additive provided in the kit (Table of Materials).</li>
			<li>Incubate the samples for 4 min at RT, then 2 min at 98 &#176;C. Afterward, samples can be used for PCR or stored at -20 &#176;C until use. Three sets of PCR reactions are used to test for the integration of the drug resistance cassette and loss of the gene of interest (Figure 4A). Refer to Table 5 for PCR reaction setup and Table 6 for thermocycler settings.</li>
		</ol>
		</li>
		<li>Identify the correct clones and transfer four clones into T25 flasks containing confluent HFFs.</li>
		<li>After individual clones lyse host cells, purify genomic DNA for further PCR verification.</li>
		<li>If an antibody recognizing the protein of interest is available, follow a standard immunoblotting procedure to verify loss of the target protein in the correct Toxoplasma knockouts. Representative images for screening a TgCPL-deletion mutant are shown in Figure 4B,C.</li>
	</ol>
	</li>
</ol>

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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nature Protocols. 8 (11), 2281-2308 (2013).</li><li>Labun, K., Montague, T. G., Gagnon, J. A., Thyme, S. B., Valen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CHOPCHOP+v2:+a+web+tool+for+the+next+generation+of+CRISPR+genome+engineering.">CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering.</a> Nucleic Acids Research. 44 (W1), W272-W276 (2016).</li><li>Heigwer, F., Kerr, G., Boutros, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-CRISP:+fast+CRISPR+target+site+identification.">E-CRISP: fast CRISPR target site identification.</a> Nature Methods. 11 (2), 2812 (2014).</li><li>Peng, D., Tarleton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EuPaGDT:+a+web+tool+tailored+to+design+CRISPR+guide+RNAs+for+eukaryotic+pathogens.">EuPaGDT: a web tool tailored to design CRISPR guide RNAs for eukaryotic pathogens.</a> Microbial Genomics. 1 (4), e000033 (2015).</li><li>Doench, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+highly+active+sgRNAs+for+CRISPR-Cas9-mediated+gene+inactivation.">Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation.</a> Nature Biotechnology. 32 (12), 1262-1267 (2014).</li><li>Sidik, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Genome-wide+CRISPR+Screen+in+Toxoplasma+Identifies+Essential+Apicomplexan+Genes.">A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes.</a> Cell. 166 (6), 1423-1435 (2016).</li></ol>

The authors have nothing to disclose.

++This protocol describes a luciferase-based protocol to assess intracellular Toxoplasma growth and evaluate the inhibition efficacy of chemical compounds against parasite growth. Compared to the existing strategies available for measuring intracellular Toxoplasma growth, this method exhibits high sensitivity and specificity. While monitoring parasite growth, a mock assay in a clear 96 well microplate is recommended to confirm that the tested strain does not prematurely lyse host cells before the end of...

Toxoplasma gondii is a highly successful obligate intracellular parasite that infects approximately one-third of the human population. Its high transmission rate is predominantly due to its diverse routes of transmission, including consumption of undercooked meat, exposure to mammalian reservoirs, and congenital transmission during birth. T. gondii mainly causes opportunistic infections that can lead to severe morbidity and mortality in immunocompromised individuals1,2,3,4,5,6. The antibiotics currently used for treating acute toxoplasmosis are particularly inefficient in treating congenital and latent infections and cause severe reactions in some individuals3,7,8. Thus, an urgent need to identify novel therapeutics exists. Understanding the differences in subcellular processes within Toxoplasma and its host will help to identify potential drug targets. Therefore, efficient and convenient genome manipulation techniques are required to study the roles of individual genes within Toxoplasma. Additionally, Toxoplasma belongs to the phylum Apicomplexa, which includes several other significant human pathogens, such as Plasmodium spp. and Cryptosporidium spp. Hence, Toxoplasma can be used as a model organism to help study basic biology in other apicomplexan parasites.
To identify novel antibiotics against microbial pathogens, high throughput screening of a library of chemical compounds is initially performed to determine their efficacy in the repression of microbial growth. So far, several microplate-based growth assays have been developed for measuring intracellular growth of T. gondii (i.e., radioactive 3H-uracil incorporation-based quantification9, quantitative ELISA-based parasite detection using T. gondii-specific antibodies10,11, reporter protein-based measurement using &#946;-galactosidase or YFP-expressing Toxoplasma strains12,13, and a recently developed high-content imaging assay14).
These individual strategies all have unique advantages; however, certain limitations also restrict their applications. For example, since Toxoplasma can only replicate within nucleated animal cells, autofluorescence and non-specific binding of anti-T. gondii antibodies to host cells cause interference in fluorescence-based measurements. Furthermore, usage of radioactive isotopes requires special safety compliance and potential safety issues. Some of these assays are more suitable for assessing growth at a single timepoint rather than continuous monitoring of growth.
Presented here is a luciferase-based protocol for the quantification of intracellular Toxoplasma growth. In a previous study, the NanoLuc luciferase gene was cloned under the Toxoplasma tubulin promoter, and this luciferase expression construct was transfected into wild-type (RH&#916;ku80&#916;hxg strain) parasites to create an RH&#916;ku80&#916;hxg::NLuc strain (referred to as RH&#916;ku80::NLuc hereafter)15. This strain served as the parental strain for intracellular growth determination and gene deletion in this study. Using the RH&#916;ku80::NLuc strain, parasite growth in human foreskin fibroblasts (HFFs) was monitored over a 96 h period post-infection to calculate parasite doubling time.
In addition, the inhibition efficacy of LHVS against parasite growth can be determined by plotting Toxoplasma growth rates against serial LHVS concentrations to identify the IC50 value. Previous literature has reported that TgCPL is a major target of LHVS in parasites and that treatment with LHVS decreases the development of acute and chronic Toxoplasma infections16,17,18,19. Additionally, RH&#916;ku80::NLuc was&#160;used as the parental strain for genome modification to generate a TgCPL-deficient strain (RH&#916;ku80&#916;cpl::NLuc), and the inhibition of LHVS was measured against this mutant. By observing an upshift of IC50 values for LHVS in the TgCPL-deficient parasites compared to the WT strain, it was validated that TgCPL is targeted by LHVS in vivo.
In this protocol, RH&#916;ku80::NLuc is used as the parental strain, which lacks an efficient non-homologous end-joining pathway (NHEJ), thereby facilitating double crossover homology-dependent recombination (HDR)20,21. Additionally, 50 bp homologous regions are flanked at both ends of a drug resistance cassette by PCR. The PCR product serves as a repair template to remove the entire gene locus via HDR using CRISPR-Cas9-based genome editing tools. Such short homologous regions can be easily incorporated into primers, providing a convenient strategy for production of the repair template. This protocol can be modified to perform universal gene deletion and endogenous gene tagging.
For instance, in our most recent publication, three protease genes, TgCPL, TgCPB (Toxoplasma cathepsin B-like protease), and TgSUB1 (Toxoplasma subtilisin-like protease 1), were genetically ablated in TgCRT (Toxoplasma chloroquine-resistance transporter)-deficient parasites using this method15. Additionally, TgAMN (a putative aminopeptidase N [TgAMN, TGGT1_221310]) was endogenously tagged15. The Lourido lab also reported using short homologous regions in the range of 40-43 bp for the introduction of site-directed gene mutation and endogenous gene tagging in the Toxoplasma genome using a similar method22. These successful genome modifications suggest that a 40-50 bp homologous region is sufficient for efficient DNA recombination in the TgKU80-deficient strain, which greatly simplifies genome manipulation in Toxoplasma gondii.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose gel extraction kit</td>
			<td>New England BioLabs</td>
			<td>T1020L</td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI</td>
			<td>New England BioLabs</td>
			<td>R0316S</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotek Synergy H1 Hybrid Multi-Mode Microplate Reader</td>
			<td>BioTek Instuments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BTX Gemini Twin Waveform Electroporation System</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemically competent E. coli cells</td>
			<td>New England BioLabs</td>
			<td>C29871</td>
			<td></td>
		</tr>
		<tr>
			<td>CloneAmp HiFi PCR premix</td>
			<td>Takara Bio</td>
			<td>639298</td>
			<td></td>
		</tr>
		<tr>
			<td>Coelenterazine h</td>
			<td>Prolume</td>
			<td>301-10 hCTZ</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRV</td>
			<td>New England BioLabs</td>
			<td>R3195S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phire Tissue Direct PCR Master Mix</td>
			<td>Thermo Scientific</td>
			<td>F170L</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid miniprep kit</td>
			<td>Zymo Research</td>
			<td>D4054</td>
			<td></td>
		</tr>
		<tr>
			<td>Q5 Site-Directed Mutagenesis kit</td>
			<td>New England BioLabs</td>
			<td>E0554S</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Geneious software for sgRNA design (version: R11)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism software (8th version)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SnapGene for molecular cloning (version: 4.2.11)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

determination of chemical inhibitor efficiency against intracellular toxoplasma gondii growth using a luciferase-based growth assay

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than &#916;cpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

Figure 1 represents an example of a growth curve for the RH&#916;ku80::NLuc strain and the derived calculation for its doubling time. Generally, the assay is performed in three technical replicates for each of the three biological replicates to account for variations of luciferase activity readings. In order to calculate the normalized fold change of parasite growth, each reading at 24-96 h post-infection was divided by the initial reading a...

Watch this Scientific Journal Video about Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay at JoVE.com

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are ...

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Immunology and Infection

7.0K Views.  Clemson University. Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

Video: Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

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 ...

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.

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.

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

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

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 ...

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 ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...