Name: determination of chemical inhibitor efficiency against intracellular toxoplasma gondii growth using a luciferase-based growth assay
Uploaded: 2020-04-29T13:30:00
Description: 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

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 thro...

<ol>
	<li>Blader, I. J., Coleman, B. I., Chen, C. T., Gubbels, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lytic+Cycle+of+Toxoplasma+gondii:+15+Years+Later.">Lytic Cycle of Toxoplasma gondii: 15 Years Later.</a> Annual Review of Microbiology. 69 (1), 1-23 (2014).</li><li>Jones, J. L., Kruszon-Moran, D., Rivera, H., Price, C., Wilkins, P. 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B., 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=An+ortholog+of+Plasmodium+falciparum+chloroquine+resistance+transporter+(PfCRT)+plays+a+key+role+in+maintaining+the+integrity+of+the+endolysosomal+system+in+Toxoplasma+gondii+to+facilitate+host+invasion.">An ortholog of Plasmodium falciparum chloroquine resistance transporter (PfCRT) plays a key role in maintaining the integrity of the endolysosomal system in Toxoplasma gondii to facilitate host invasion.</a> PLOS Pathogens. 15 (6), e1007775 (2019).</li><li>Larson, E. 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++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 ...

These sensitive strategies can be used to evaluate the inhibition efficacy of chemical inhibitors and in combination with CRISPR-Cas9 gene deletion to investigate the drug targets. Instead of using an endpoint assay, parasite growth can be monitored over time to allow calculation of parasite doubling time using a sensitive reporter protein. This assay can potentially be scaled up in 384 or 1, 536 well microplates for the testing of multiple compounds or for the screening of libraries in a high throughput manner.This strategy can be modified to quantify the growth of other intracellular microbial pathogens and their responses to the drug of interest. To perform a luciferase-based Toxoplasma growth assay, begin by carefully aspirating the medium from pre-seeded 96-well microplate cultures of human foreskin fibroblasts and inoculate 150 microliters of parasite B suspension into the wells in a three column-five row format. After a four-hour incubation in the cell culture incubator, carefully aspirate the medium from each well to remove the non-invaded parasites and fill the wells in each but the first row with room temperature phenol red free medium.Then add 100 microliters of a 12.5 micromolar luciferase substrate solution in PBS into each well of the top row and incubate the microplates for 10 minutes at room temperature to allow full cell lysis. At the end of the incubation, measure the luciferase activity on a microplate reader. Each reading represents the initial number of invaded parasites at four hours post infection.Repeat the analysis every 24 hours for the next four days without changing the medium. To calculate the normalized fold-change of parasite growth, each reading can be divided by the initial reading at four hours post infection. The log 2 of the normalized fold-change of parasite growth can then be plotted against each time point and subjected to a linear regression function to obtain the slope which represents the doubling time.To assess the efficacy of the inhibition of a compound of interest on Toxoplasma growth, culture human foreskin fibroblasts in 96-well microplates for at least seven days before inoculating each well of cells with 150 microliters of parasites as demonstrated for four hours at 37 degrees Celsius and 5%carbon dioxide. During the incubation, prepare the compound of interest at eight different concentrations in a 12-well reservoir by serial dilution. Dilute the compounds in a 12-well reservoir at a one-to-three dilution by pipetting each mixture up and down several times with a one milliliter pipette.At four hours post infection, replace the medium in each well from columns two through nine with 150 microliters of medium supplemented with each dilution of compound, leaving the first column filled with regular medium to serve as the non-treated control. Then return the cell cultures to the cell culture incubator for 96 hours and measure the luciferase activity for each well to determine the percentage of growth inhibition in the cultures at each concentration of compound. To calculate the normalized luciferase activity as a percentage, divide the average luciferase activity for each compound concentration by the average luciferase activity derived from the non-treated parasites.Then use an appropriate graphing software program to plot the normalized luciferase activities against the individual compound and to calculate the half maximal inhibitory concentration for each compound. To generate a plasmid construct expressing single-guide RNA and Cas9 for deleting a gene of interest, navigate to the Toxoplasma Genomics Resource page and retrieve the entire gene coding sequence including the introns and exons along with the 1.5 kilobase five and three prime untranslated regions. Copy the retrieved TgCPL sequence into the sequence analysis software and label the five and three prime untranslated regions.On the Toxoplasma Genomics Resource page, click Download, Data Files, and Current_Release. In the T.gondii GT1 folder, select FASTA. From the data folder, download the Toxoplasma gondii GT1 strain genome sequence file.Then create a local folder named Toxoplasma Genome in the DNA sequence analysis software and import the Toxoplasma genome sequence file into the folder. Select Tools, Cloning, and Find CRISPR Sites and set three prime Cas9 prime for the PAM site location. Select a single-guide RNA that demonstrates a high specificity score, generally greater than 98%and lacks a G following the NGG.The selected single-guide RNA is usually located at sites close to the start and stop codons of the gene of interest. Then use a PCR premix with the settings as indicated in the table to perform a PCR reaction to modify the preexisting plasmid expressing single-guide RNA that targets the TgUPRT gene. For Toxoplasma transfection, mix two micrograms of repaired template DNA with 20 micrograms of the single-guide RNA Cas9 expression plasmids and mix 400 microliters of parasite resuspension, DNA, and five microliters of 200 millimolar ATP 500 millimolar reduced glutathione in a 1.5 milliliter centrifuge tube.Add cytomix buffer to bring the total volume up to 500 microliters as necessary and transfer the parasite DNA mixture to a four millimeter gap with electroporation cuvette. Then electroporate the parasites at two kilovolts and 50 ohms of resistance. To clone knockout parasites, first perform a two-step dilution of the parasites to achieve a 10 parasites per milliliter of D10 medium supplemented with the appropriate antibiotic concentration.Next, transfer 150 microliters of the resuspended purified parasites to each well of a 96-well microplate pre-seeded with foreskin fibroblasts and incubate the microplate at 37 degrees Celsius and 5%carbon dioxide for seven days. At the end of the incubation, identify the wells containing a single plaque and transfer 75 microliters of the resuspended Toxoplasma-infected human foreskin fibroblasts from each well of the 96-well microplate culture into individual 1.5 milliliter tubes. Collect the cells by centrifugation and resuspend the pellets in 10.25 microliters of lysis buffer containing dilution buffer in DNA release additive.Then incubate the samples for four minutes at room temperature and two minutes at 98 degrees Celsius before performing PCR to test for the integration of the drug resistance cassette and loss of the gene of interest. The inhibition efficacies of LHVS and wild type in delta CPL strains can be determined by plotting their luciferase activities against different inhibitor concentrations. After PCR, the mutated plasmid is linearized and loaded into a 1%agarose gel for the verification of a successful amplification.After gel extraction and E.coli transformation, the clones containing the expected plasmids can be screened by restriction endonuclease digestion in DNA sequencing. After repair template amplification, agarose gel electrophoresis can be used to verify the correct size of the PCR product. Generally, seven to eight clones are selected for the initial screening to check for deletion of the coding sequence of the gene of interest, followed by detection of the five and three prime arms to help minimalize the total number of clones to be screened.Further verification can be completed by immunoblotting if any antibody recognizing the target protein is available. Before obtaining luciferase activity measurements, a clear microplate with a mock infection should be checked under a microscope to ensure that the parasites do not fully lyse the host cells.

determination-chemical-inhibitor-efficiency-against-intracellular

Research

JoVE Journal

Immunology and Infection

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).

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-&#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 ...