Special thanks to Dr. Peter Bradley for the antibody to detect the T. gondii mitochondria. The work was supported by National Institute of Health Grants AI072028 and AI107431 to D.G.M and a generous donation to New York Medical College for the study of tropical medicine.

The authors have no competing financial interests.

The described protocol provides a non-biased approach that uses activation of murine bone marrow-derived macrophages and forward genetics to isolate T. gondii mutants with a defect in their ability to survive activation of infected macrophages. The phenotype of the mutants following macrophage activation typically falls into one of two broad categories: 1) The parasites appear intact but fail to replicate beyond 1 parasite per PV; 2) The parasites appear degraded and may be in spacious, amorphous PVs. The fact that the mutants have a phenotype like wild type parasites in na&#239;ve macrophages in the absence of activation proves the protocol can specifically be used to identify mutants that are specifically impaired in their ability to resist macrophage activation. The use of primary bone marrow-derived macrophages is recommended versus the RAW 264.7 macrophage cell line for the screen because the morphology of the parasites and parasite number per vacuole are more easily visualized in bone marrow-derived macrophages.
The described screen combined with forward genetics provides a versatile tool to dissect parasite genes and ultimately genetic pathways important for resistance to specific mediators of cell autonomous immunity. Such forward genetics studies are important to identify parasite genes that can be followed like a string to begin unravelling parasite pathways important for resisting specific antimicrobial mediators both in vitro and during pathogenesis. A previous difficulty with forward genetic approaches was identification of the key gene disrupted in each mutant. Cosmid library complementation in T. gondii has been quite effective for functional complementation of cell division mutants. However, the fact that replication of wild type parasites are also suppressed by IFN-&#947; and LPS just to a lesser extent than the mutants, makes functional complementation more difficult than for cell division mutants. Whole genome mutational profiling for both chemical and insertional mutants in T. gondii has recently emerged as a productive, and even cost effective, avenue to identify the genes responsible for phenotypes of mutants in forward genetics screens 34,36,37,44. Consequently, whole genome mutational profiling using next generation sequencing has enhanced the potential uses of chemical mutagenesis of T. gondii in forward genetic studies to identify genes important for specific functions. Such a forward genetics approach as described in the current protocol are important as most of the genome of T. gondii remains hypothetical with a majority of predicted genes having no homology to other genes or functional domains that might aid in the identification of candidate parasite genes important for immune evasion.
IFA analysis of 96 well plates is not generally considered a high throughput screening method. In our experience it is reasonable for one person to stain and screen 10 96-well plates in a day or approximately 960 mutants. In the paper by Skariah et al., approximately 8,000 mutants were analyzed and 14 independent mutants were isolated that were significantly impaired for survival/replication in activated but not na&#239;ve macrophages 28. The impaired survival of the mutants was predominately the result of increased susceptibility to nitric oxide. The limitation in the overall method is primarily at the level of obtaining single clones of the parasite rather than screening by microscopy as the initial screen in 96 well plates is qualitative and reasonably rapid. Confirmation of the phenotype of mutants identified in the screen of the 96 well plate is followed up in Step 3 by quantitative and qualitative analysis using macrophages in chamber slides and by adding equal numbers of wild type or mutant parasites. The use of other analytical screening methods such as fluorometry at the total population level of parasites, rather than the individual parasite level as evaluated by microscopy, are problematic in the current protocol as many of the degraded parasites stain with the polyclonal antibody against T. gondii as robustly as wild type parasites with the difference in the mutant being more qualitative than quantitative at the level of fluoresence intensity. Also, the dose of IFN-&#947; and LPS required to isolate mutants with a defect in resistance to activation of infected macrophages results in suppressed replication of wild type parasites albeit at later time points. Therefore, measurement of total parasite number for the forward genetics screen including the use of luminescent parasites is problematic. However, it is possible the staining protocol could be eliminated in the screening step if the parental parasite clones used for chemical or insertional mutagenesis expressed a constitutive or inducible fluorescent or luciferase marker. The described protocol using IFA and microscopy screening of macrophages in 96 well plates allows for isolation of mutants defective for survival/replication in activated macrophages but it also clearly misses some potential mutants due to the limited microscopic resolution achievable using the 96-well plate format as at this resolution amorphous swollen vacuoles that stain for parasite antigen may be mistaken for healthy replicating parasites within a normal PV. Substitution of 96 well cover glass plates, instead of 96 well plates with an optical surface, is likely to achieve greater resolution during the screen of infected macrophages in 96 well plates.
The vast majority of the mutants identified using IFN-&#947; and LPS in the described protocol to activate macrophages following parasite invasion have a defect in their ability to protect themselves from nitric oxide 28. In this regard, the screen although intended to be non-biased, may enrich for the isolation of parasite mutants with increased susceptibility to specific antimicrobial mediators depending on the activation conditions, the type and species of macrophages and the timing of parasite invasion relative to macrophage activation that are chosen for the screen. Thus the innate immune cell chosen for the screen and the activation stimuli applied are critical parameters that impact the type of antimicrobial mediators to which the resultant parasite mutants are likely to be susceptible. The genotype of the parasite is also a critical parameter as Type I genotypes of the parasite are relatively resistant to immunity related GTPases (IRGs) induced in response to IFN-&#947; while the Type II and III genotypes are more susceptible 26,45,46. In the described protocol, macrophages are activated after parasite invasion. Although the Type II genotype, Prugnaud strain, used in the described screen is susceptible to the action of immunity related GTPases, allowing the parasite to invade macrophages first enables replication of wild type parasites before full induction of the IRGs. Also, it is not clear that IRGs are effective against the parasite if they are induced in macrophages subsequent to parasite invasion and initiation of replication.
The described protocol uses macrophages to identify parasite genes important for resistance to IFN-&#947;-dependent mediators of cell autonomous immunity, particularly nitric oxide. The isolation of a pool of parasite mutants that share a marked susceptibility to nitric oxide as well as a shared phenotype of nitric oxide-dependent mitochondrial fragmentation provides a unique set of tools to identify parasite genes and pathways important for resistance to nitric oxide and for understanding the action of nitric oxide more broadly against T. gondii and other eukaryotic pathogens.The described protocol with minor modifications can be used for forward genetics studies to identify parasite genes important for resistance to different mediators of cell autonomous immunity. Potential modifications include altering the type of host cell infected, the activation stimuli, or the timing of activation relative to parasite invasion. For example, pre-activation of murine macrophages with IFN-&#947; before addition of parasites would result in activation of immunity related GTPases. Such a model could be used to identify parasite genes in Type I genotypes of T. gondii that may contribute to the resistance mediated by ROP18 and ROP5 to immunity related GTPases 24,25. Mediators of cell autonomous immunity against T. gondii in human innate immune cells are not as well defined as in rodents. Thus, the use of human peripheral blood monocytes to screen chemical mutants of T. gondii might identify both parasite genes important for survival during human infection in innate immune cells as well as mechanisms important in humans for antimicrobial resistance to T. gondii. Parasite genes important for resistance to specific antimicrobial mediators can be identified by isolating mutants unable to survive exposure of infected host cells to pharmacological agents such as reactive oxygen or nitrogen species. Similarly the protocol can be modified to isolate parasite mutants with increased susceptibility to inflammasome activation 47-49 or ATP stimulation of the macrophage purinergic receptors 50.
Taken together, the protocols describe an approach using forward genetics and innate immune cells to isolate parasite mutants important for T. gondii resistance to IFN-&#947;-dependent cell autonomous immunity. Importantly, the approach put forth is versatile and can be readily modified to isolate parasite genes important for evading specific antimicrobial mediators, parasite survival in specific types of host cells or resistance to stress conditions encountered during toxoplasmosis. Furthermore, parasite genes important for resisting host cell activation in many cases may also play a role directly or indirectly in cyst production in vivo during infection.

Toxoplasma gondii (T. gondii) is an obligate intracellular, protozoal pathogen. It is the causative agent of toxoplasmosis, a health hazard in immunocompromised individuals. It is also the model system for other apicomplexan pathogens that infect humans including Cryptosporidium and Cyclospora. Toxoplasmosis is most commonly acquired through ingestion of food or water contaminated with the bradyzoite or oocyst stage of the parasite. Upon ingestion, these stages convert to the tachyzoite stage of the parasite that replicates within host cells and disseminates systemically. T cells, IFN-&#947; and, to a lesser extent, nitric oxide 1-4, are important for control of infection but are not capable of eliminating the disease, as a proportion of tachyzoites convert to the bradyzoite stage that are protected within tissue cysts resulting in a long-lived chronic infection. In fact, there are no therapeutics effective against the chronic cyst stage of the disease. Severe toxoplasmosis is most often due to the reactivation of persistent infection, with the bradyzoite stage of the parasite converting back to the rapidly replicating tachyzoite stage characteristic of primary and acute infection.
Early survival in the face of the innate immune response is important to allow the parasite to reach sufficient parasite numbers, as well as to reach distal sites, to enable establishment of chronic infection. T. gondii has evolved strategies to counteract host defense mechanisms that likely contribute to its ability to replicate and disseminate early in infection. First, T. gondii forms a unique PV during parasite invasion that is largely segregated from the endocytic and exocytic processes of the host cell compared to other intracellular pathogens 5-9. Also, like all successful intracellular pathogens T. gondii modifies its host cell to create a permissive environment for growth. This includes reprogramming host cell gene expression by altering host cell transcription factors including those important for regulating cell activation 10-15. ROP16 16-19, GRA15 20, GRA16 21 and GRA24 22 have all been shown to be important in regulating the transcriptional response and cell signaling cascades of host cells infected with T. gondii. Recent studies using genetic crosses between parasite strains with distinct phenotypes have been highly productive in identifying parasite genes that underlie parasite genotype-dependent traits including evasion of immunity related GTPases (IRGs) 16,19,23-26. In mice, immunity related GTPases (IRGs) are critical for the control of Type II and III genotypes of the parasite while the very virulent Type I genotypes have evolved mechanisms to evade the murine IRGs. However, it is also clear that the parasite has evolved mechanisms to evade antimicrobial mediators in addition to the IRGs and that some of these mechanisms may be conserved across parasite genotypes 27,28. In addition, very little is known about the critical mediators of cell autonomous immunity against T. gondii during human toxoplasmosis. Parasite genes important for resistance to mediators of cell autonomous immunity may also be important for survival during tachyzoite to bradyzoite conversion which can also be triggered by host immune responses. For example, nitric oxide at high levels can suppress parasite replication in infected macrophages but it can also stimulate tachyzoite to bradyzoite conversion resulting in cyst production30-32.
ToxoDB is a functional genomic database for T. gondii that functions as a critical resource for the field in terms of providing sequence information for the parasite genome and access to published and unpublished genomic scale data including community annotations, gene expression and proteomics data 33. Similar to many protozoal pathogens, the majority of the genome consists of hypothetical genes with no information available based on gene homology to provide insight into their potential functions. Thus, forward genetics is a powerful tool to identify novel parasite genes important for immune evasion, cyst conversion and other functions critical for parasite pathogenesis as well as for conversion between distinct developmental stages. An additional strength of forward genetics is that it can be used as a relatively non-biased approach to interrogate the parasite as to the genes that are important for specific tasks in pathogenesis, including immune evasion and cyst formation. Recent improvements in next generation sequencing for mutational profiling have made it a method of choice for identifying the responsible parasite genes from forward genetics studies using both chemical and insertional mutagenesis 34-37.
It is important to identify vulnerabilities in T. gondii that can be exploited to enhance the effectiveness of cell autonomous immune mechanisms against the parasite particularly those that may also be active against the resistant cyst stage. Toward this aim, an in vitro murine macrophage infection and activation model was developed to identify mutations in the parasite that specifically impair T. gondii fitness following activation of infected macrophages but not in na&#239;ve macrophages. This macrophage screen was used to interrogate a library of T. gondii insertional mutants in order to ultimately identify T. gondii genes important for resistance to nitric oxide 27,28. The isolation of a panel of T. gondii mutants with impaired resistance to activation of infected macrophages, particularly a marked sensitivity to nitric oxide, proved the utility of the screen to identify parasite genes important for resistance to mediators of cell autonomous immunity other than the resistance mechanisms described for the murine IRGs 28. Insertional mutagenesis has advantages over chemical mutagenesis in terms of generating a limited number of random mutations in each parasite clone and, in theory, easier identification of the site of mutation. However, identifying the genomic site of plasmid insertion in T. gondii insertional mutants, in practice, has been surprisingly difficult in many cases 37. Insertion of a plasmid into a gene is also likely to disrupt the function of a gene in contrast to chemical mutagenesis that typically results in single nucleotide changes. However, chemical mutagenesis with either N-ethyl-N-nitrosourea (ENU) or ethylmethane sulfonate (EMS) may offer an increased ability to analyze a larger portion of the parasite genome, compared to insertional mutagenesis, as it creates multiple single nucleotide polymorphisms (estimated at 10 -100) per mutant34,38. Moreover, recent advances in whole genome profiling has made it possible to use next generation sequencing to identify the most likely candidate genes responsible for the identified phenotype of a mutated parasite 34,38. Regardless of the mutagenesis approach, confirmation of the role of the parasite gene in resistance to macrophage activation ultimately requires gene deletion and complementation to fulfill molecular Koch&#8217;s postulates.
The ability to dissect the function of a gene by genetic manipulation of both the parasite and the macrophage is important as many of the genes identified via forward genetics in T. gondii, as well as other pathogens, are still characterized as hypothetical genes with little to no sequence homology to other proteins with known functions. The current paper outlines a general approach that can be used to identify whether the disrupted gene in a mutant is important for resistance to a known or unknown mediator of cell autonomous immunity. The initial analysis of host antimicrobial factors is performed by evaluating the survival of wild type and mutant parasites in macrophages from wild type mice versus those with specific gene deletions in inducible nitric oxide synthase (iNOS), gp-91 phox (NADPH oxidase), and specific immunity related GTPases (IRGs). This will determine if the identified parasite genes are important for resistance to nitric oxide, reactive oxygen intermediates or immunity related GTPases 28 respectively or if an unknown immune mechanism is involved. Activation of infected macrophages with both IFN-&#947; and LPS, described in the current protocol, results primarily in the isolation of parasite genes important for resistance to nitric oxide 28. The use of pharmacological agents that induce nitric oxide in the absence of macrophage activation (nitric oxide donors) confirmed that the majority of the genes identified were important for resistance to nitric oxide rather than nitric oxide in concert with additional mediators associated with macrophage activation 28.
Step one and two describe a forward genetics screen designed to isolate parasite mutants with a fitness defect following activation of infected bone marrow-derived macrophages in vitro. Step one describes a dose titration analysis to empirically determine a dose of IFN- &#947;&#160; and LPS to use for macrophage activation that reduces parasite replication but does not fully inhibit replication of the wild type T. gondii parental strain that is used for creation of the library of parasite mutants. Step two describes the forward genetic screen of the mutant clones in macrophages in 96-well plates. Step three outlines an approach to confirm the phenotype of each mutant identified in the screen of the 96 well plates and to evaluate whether the defect in each mutant affects parasite survival, replication, or cyst production in response to macrophage activation. Step four describes the use of bone marrow-derived macrophages from mice with deletions in specific antimicrobial pathways to identify the immune mediators to which the parasite mutant is specifically susceptible. Step five outlines an approach to determine if a parasite mutant is also compromised for in vivo pathogenesis as evaluated by cyst production in the brains of infected mice.

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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.

Toxoplasma gondii replicates freely in na&#239;ve macrophages and has a doubling time between 6-12 hr&#160;depending on the strain of the parasite. Figure 1 shows representative parasites in na&#239;ve versus activated bone marrow-derived macrophages. Figure 2 shows the general morphology of parasites in HFF host cells at 2, 4, 8, 16 and 32 parasites/PV. In the current protocol, the parasite is allowed to invade na&#239;ve macrophages and establish a nascent parasitophorous vacuole (PV) prior to the delivery of a potent activation stimulus, LPS and IFN-, to the infected macrophages. In this model, replication of wild type parasites is slowed, but parasite replication still proceeds for approximately 24 hr during which time the macrophages become progressively more adept at inhibiting parasite replication. The screen is designed to function as a modified competition model between the time required for potent macrophage activation versus the time during which the wild type parasite or parasite mutant can continue to replicate within its PV. The first step in the screen is to choose a dose of LPS and IFN- &#947; to be used for activation of infected macrophages. The dose is empirically determined by evaluating parasite replication in macrophages activated with either a constant dose of IFN- &#947; in combination with a range of LPS concentrations or a constant dose of LPS with a range of IFN- concentrations (Step 1). The dose of the activation stimuli needs to be evaluated for the parental wild type parasites used for creation of the parasite mutants by chemical or insertional mutagenesis. Ideally the dose of LPS and IFN- &#947; will permit parasite replication to an average of 2-8 parasites per PV 24-30 hr after activation compared to 8-16 parasites per PV in na&#239;ve macrophages (Figures 1 and 2).
Once the appropriate concentration of LPS and IFN- &#947; is determined, the mutants are screened in 96-well optical bottom tissue culture plates. The optical bottom in the plates is important because irregularities in the plastic that is used for traditional tissue culture plates makes it difficult to achieve the appropriate resolution to screen the parasites by phase contrast and fluorescence microscopy. Phase contrast and fluorescence microscopy of the 96-well plates also requires an inverted fluorescence microscope with phase contrast 4, 10, 20 or 40x objective equipped for long working distances. Mutants are screened in replicate plates containing murine bone marrow-derived macrophages. After challenge with parasite mutants, infected macrophages in the wells of one plate are activated with LPS and IFN- &#947; while the other plate containing infected macrophages is cultured in only D10 media. Plates are incubated at 37 &#176; C and 5% CO2 for 24-30 hr after addition of LPS and IFN- &#947; . After incubation, cells are fixed, stained for parasites and analyzed by IFA using both fluorescence and phase contrast microscopy. Mutants are selected that have normal parasite numbers and replication in na&#239;ve macrophages but fewer parasites or smaller vacuoles with fewer parasites in infected macrophages that are activated. Once mutants are selected they should be rescreened in chamber slides (Step 3) in na&#239;ve and activated macrophages in two independent experiments to enable analysis of parasite morphology at higher magnifications (100X objective and oil) and to confirm that they have normal replication in na&#239;ve macrophages but a defect in replication/survival following macrophage activation.
The phenotypes of the mutants with defects in resistance to activation of infected macrophages typically fall into two broad categories: parasites that appear morphologically intact but are unable to replicate beyond a single parasite per PV; and parasites that appear to be degraded and may also be in amorphous spacious PVs (Figures 1 and 3). The morphology and ultrastructure of the parasite and PV is best viewed on slides using a 100X objective and oil and by phase contrast microscopy combined with fluorescence analysis. Staining of parasites for IFA with an antibody to lysosomal associated membrane protein-1 (LAMP1) is useful to determine whether the defect in the mutant is associated with an inability of the PV to prevent fusion with lysosomes. Shown in Figure 4 is a parasite within a phagolysosome (LAMP1 positive) versus a parasite that is in a PV that is largely segregated from the endocytic system of the host cell (LAMP1 negative). The phagolysosome is evident by a continuous solid rim of LAMP1 straining around the entire parasite. In contrast, a PV often has lysosome organelles in the vicinity but no continuous rim of LAMP1 staining around its circumference. The use of antibodies to defined structures/organelles within the parasite and/or PV are useful in follow up IFA studies to identify anomalies in each mutant associated with activation of infected macrophages. Such interrogations with antibodies may provide insight into the mechanisms that underlie the replication/survival defect in a mutant. For example, Figure 5 shows the T. gondii mitochondrion stained with monoclonal antibody 5F4 anti-F1 ATPase beta subunit (a kind gift from Peter Bradley) 24 hr after activation of macrophages infected with wild type parasites or parasite mutants. T. gondii was co-stained with a monoclonal anti-T. gondii antibody. Wild type T. gondii has a single mitochondrion that extends around the circumference of the parasite. The parasite&#8217;s sole mitochondrion in wild type parasites following activation of infected macrophages was intact compared to a fragmented mitochondrion in mutant parasites. Mitochondria fragmentation was evident not only in degraded/amorphous parasites but also in parasites that displayed normal morphology as evaluated by phase contrast microscopy. This suggests that the defect in the parasite mutant may increase the susceptibility of the parasite mitochondrion to host cell mediators induced by macrophage activation.
The current model uses a combination of IFN- &#947; and LPS to activate infected macrophages. IFN- &#947; stimulates the transcription factor STAT1. LPS, like TNF-&#945;, stimulates the transcription factor NF-&#954;B. Known IFN- &#947; -dependent antimicrobial mediators important in cell autonomous immunity against T. gondii include an array of IFN- &#947; -dependent immunity related GTPases (IRGs, GBPs) and reactive nitrogen and oxygen species in addition to other agents. In order to determine if the defect in each mutant is specifically associated with production of known IFN- &#947; -inducible antimicrobial mediators, bone marrow-derived macrophages are isolated from iNOS-/-, gp 91 phox-/- or specific IRG/GBP gene deleted mice and assayed for activity against the mutant parasites. The combination of IFN- &#947; and LPS to activate infected macrophages preferentially results in mutants with enhanced susceptibility to nitric oxide compared to wild type parasites 28.
Activation of infected macrophages can induce stage differentiation of the parasites from tachyzoites to bradyzoites that are contained in tissue cysts. Such cysts are characteristic of chronic infection. Thus, parasite mutants that are defective for replication/survival following activation of infected macrophages may also be defective for conversion to cysts during in vivo infection. In order to evaluate cyst production during chronic infection, mice are challenged intraperitoneally (ip) with a non-lethal dose of the parental parasite strain or the mutant clone. Cysts in the brain range in size from less than 10 &#956;m in diameter to greater than 50 &#956;m as shown in Figure 6. Count the number of both large and small cysts by IFA but keep the counts separate in order to determine if the parasite mutant is impaired for total cyst production or just for establishment and maintenance of large cysts.
<img alt="Figure 1" src="/files/ftp_upload/52556/52556fig1.jpg" /> 
Figure 1. Na&#239;ve murine bone marrow-derived macrophages are permissive for replication of T. gondii&#160;but activation with IFN- &#947; and LPS substantially inhibits replication. (A)&#160;A parasitophorous vacuole (PV) of wild type T. gondii 24 hr after invasion of na&#239;ve murine bone marrow-derived macrophages. (B)&#160;A PV of wild type T. gondii parasites consisting of four parasites per vacuole 24 hr after activation of infected macrophages. (C)&#160;An example of a mutant parasite unable to replicate and still at one parasite/PV 24 hr after activation of infected macrophages. (D)&#160;An example of a mutant parasite that appears degraded within an amorphous PV 24 hr after activation of infected macrophages. The top row shows phase images of the parasites and the bottom row shows fluorescent images using a polyclonal anti-serum against T. gondii. The scale bar represents 5 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52556/52556fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52556/52556fig2.jpg" /> 
Figure 2. Parasite replication evaluated by the number of parasites per PV. The pictures show 2, 4, 8, 16 and 32 parasites per PV in HFF cells. Each picture is a merged phase image that shows polyclonal antibody staining of the parasite in red and the HFF nucleus in blue (DAPI). The scale bar represents 5 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52556/52556fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52556/52556fig3.jpg" /> 
Figure 3. Mutants exhibit a range of phenotypes following activation of infected macrophages. The column on the left is a phase image of the parasite in macrophages, the center column is a fluorescent image using an antibody to T. gondii, and the right column is a merge of the phase and fluorescent image. The parasite is shown in green and the macrophage nucleus in blue. The scale bar represents 5 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52556/52556fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52556/52556fig4.jpg" /> 
Figure 4. LAMP1 staining distinguishes PVs from phagolysomes-containing parasites. Parasites are stained with a polyclonal anti-T. gondii antibody (red) and LAMP1 with the mAb 1D4B (green). An arrow marks a parasite PV that has fused with lysosomes. The parasite without the arrow is in a parasitophorous vacuole (PV) that has not fused with lysosomes. The scale bar represents 5 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52556/52556fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52556/52556fig5.jpg" /> 
Figure 5. Wild type parasites maintain an intact mitochondrion while the mitochondrion is degraded in parasite mutants following activation of infected macrophages. Mitochondrion (green) in wild type parasites (top panel) compared to three different mutant T. gondii parasites at different stages of degradation (bottom three panels) 24 hr after activation of infected macrophages. The scale bar represents 5 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52556/52556fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52556/52556fig6.jpg" /> 
Figure 6. Detection of parasite cysts in the brain using FITC-conjugated dolichos biflorus lectin. The cyst wall labeled with the lectin is shown in green. The scale bar represents 50 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52556/52556fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-Î³-Dependent Cell Autonomous Immunity at JoVE

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-γ Dependent Cell Autonomous Immunity | Immunology and Infection | Video

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.

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 ...

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11.0K Views.  New York Medical College. 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. 

Video: Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-γ-Dependent Cell Autonomous Immunity

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 ...

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 ...

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 ...

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 ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8+ T proliferation and to promote CD4+Foxp3+ Treg expansion in vitro.

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Medicine

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.

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.

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

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-γ-Dependent Cell Autonomous Immunity

Summary

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

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

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

Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

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