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><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 &lt;em&gt;E. coli&lt;/em&gt; 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>&lt;strong&gt;Software&lt;/strong&gt;</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 (8&lt;sup&gt;th&lt;/sup&gt; 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 ...

determination-chemical-inhibitor-efficiency-against-intracellular

Research

JoVE Journal

Immunology and Infection

7.1K Views.  Clemson University. Deze gevoelige strategieën kunnen worden gebruikt om de remmingseffectie van chemische remmers te evalueren en in combinatie met CRISPR-Cas9-gendeling om de medicijndoelen te onderzoeken. In plaats van een endpoint-test te gebruiken, kan de groei van parasieten na verloop van tijd worden gecontroleerd om de berekening van de verdubbelingstijd van parasieten mogelijk te maken met behulp van een gevoelig reportereiwit. Deze test kan mogelijk worden opgeschaald in 384 of 1, 536 goed microplaten...

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

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Details are presented on how QTL mapping with a whole genome sequence based genetic map can be used to identify a drug resistance gene in Toxoplasma gondii and how this can be verified with the CRISPR/Cas9 system that efficiently edits a genomic target, in this case the drug resistance gene.

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

Genetics

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 ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

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-&#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 ...

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive patients, organ transplant recipients and pregnant women, resulting in the severe health condition, Toxoplasmosis. Another major protozoan, Neospora caninum, which bears many similarities to Toxoplasma gondii, causes serious diseases in animals, as does Encephalomyelitis and Myositis-Polyradiculitis in dogs and cows, resulting in stillborn calves. All these exhibited similar nucleoside triphosphate hydrolases (NTPase). Neospora caninum has a NcNTPase, while Toxoplasma gondii has a TgNTPase-I. The enzymes are thought to play crucial roles in propagation and survival. In order to establish compounds and/or extracts preventing protozoan infection, we targeted these enzymes for drug discovery. The next step was to establish a novel, highly sensitive, and highly accurate assay by combining a conventional biochemical enzyme assay with a fluorescent assay to determine ADP content. We also validated that the novel assay fulfills the criteria to carry out high-throughput screening (HTS) in the two protozoan enzymes. We performed HTS, identified 19 compounds and six extracts from two synthetic compound libraries and an extract library derived from marine bacteria, respectively. In this study, a detailed explanation has been introduced on how to carry out HTS, including information about the preparation of reagents, devices, robot arm, etc.

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Toxoplasma gondii and Neospora caninum infections are found in humans and animals and lead to serious health issues. The two parasites share similar nucleoside triphosphate hydrolases and play important roles in propagation and survival. We established a high-standard assay of the enzymes requiring robot arm usage.

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive ...

Biochemistry

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing &#946;-galactosidase

Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many non-endemic countries due to the increase in migration. This disease affects nearly 8 million people, with new cases estimated at 50,000 per year. In the 1960s and 70s, two drugs for ChD treatment were introduced: nifurtimox and benznidazole (BZN). Both are effective in newborns and during the acute phase of the disease but not in the chronic phase, and their use is associated with important side effects. These facts underscore the urgent need to intensify the search for new drugs against T. cruzi.
T. cruzi is transmitted through hematophagous insect vectors of the Reduviidae and Hemiptera families. Once in the mammalian host, it multiplies intracellularly as the non-flagellated amastigote form and differentiates into the trypomastigote, the bloodstream non-replicative infective form. Inside the insect vector, trypomastigotes transform into the epimastigote stage and multiply through binary fission.
This paper describes an assay based on measuring the activity of the cytoplasmic &#946;-galactosidase released into the culture due to parasites lysis by using the substrate, chlorophenol red &#946;-D-galactopyranoside (CPRG). For this, the T. cruzi Dm28c strain was transfected with a &#946;-galactosidase-overexpressing plasmid and used for in vitro pharmacological screening in epimastigote, trypomastigote, and amastigote stages. This paper also describes how to measure the enzymatic activity in cultured epimastigotes, infected Vero cells with amastigotes, and trypomastigotes released from the cultured cells using the reference drug, benznidazole, as an example. This colorimetric assay is easily performed and can be scaled to a high-throughput format and applied to other T. cruzi strains.

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing &#946;-galactosidase

We describe a high-throughput colorimetric assay measuring &#946;-galactosidase activity in three life cycle stages of Trypanosoma cruzi, the causative agent of Chagas disease. This assay can be used to identify trypanocidal compounds in an easy, fast, and reproducible manner.

Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Luciferase-Based Growth Assay to Quantify Intracellular Parasite Reproduction

Source: Key, M., et al., <a href="https://app.jove.com/v/60985">Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay.</a> J. Vis. Exp. (2020)
In this video, we demonstrate the luciferase assay to quantify the growth of the intracellular pathogen Toxoplasma gondii. Utilizing a strain of Toxoplasma that expresses luciferase enzyme after infecting the host cells, the luciferase activity is detected in the infected cell culture to determine the successful reproduction of the parasite.

Source: Key, M., et al., Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth ...

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Biochemical assays

Luciferase Assay to Study Efficacy of Antiparasitic Chemicals Against Parasites

Source: Key, M., et al. <a href="https://app.jove.com/v/60985">Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay.</a> J. Vis. Exp. (2020).
The video demonstrates the luciferase assay used to determine the efficacy of an antiparasitic chemical inhibitor against the intracellular pathogen Toxoplasma gondii. The study involves luciferase-expressing parasites invading the host cells to replicate and survive. The chemical inhibitor binds with the cysteine proteases of the parasite and inhibits replication. The luciferase activity detected is proportional to the fold changes in parasite growth, determining the drug&#39;s efficacy against the parasite.

Source: Key, M., et al. Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth ...

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

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