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Calcium dependent protein kinase 1 (CDPK1) is an essential Ser/Thr kinase that controls invasion and egress by the protozoan parasite Toxoplasma gondii . The Gly gatekeeper of CDPK1 makes it exquisitely sensitive to inhibition by small molecule 1 H -pyrazolo[3,4- d ]pyrimidine-4-amine (PP) compounds that are bulky ATP mimetics. Here we rationally designed, synthesized, and tested a series of novel PP analogs that were evaluated for inhibition of CDPK1 enzyme activity in vitro and parasite growth in cell culture. Optimal substitution on the PP scaffold included 2-pyridyl ethers directed into the hydrophobic pocket and small carbocyclic rings accessing the ribose-binding pocket. Further optimization of the series led to identification of the lead compound 3a that displayed excellent potency, selectivity, safety profile, and efficacy in vivo . The results of these studies provide a foundation for further work to optimize CDPK1 inhibitors for the treatment of acute and chronic toxoplasmosis.

Cryptosporidium parvum and Cryptosporidium hominis have emerged as major enteric pathogens of infants in the developing world, in addition to their known importance in immunocompromised adults. Although there has been recent progress in identifying new small molecules that inhibit Cryptosporidium sp. growth in vitro or in animal models, we lack information about their mechanism of action, potency across the life cycle, and cidal versus static activities. Here, we explored four potent classes of compounds that include inhibitors that likely target phosphatidylinositol 4 kinase (PI4K), phenylalanine-tRNA synthetase (PheRS), and several potent inhibitors with unknown mechanisms of action. We utilized monoclonal antibodies and gene expression probes for staging life cycle development to define the timing of when inhibitors were active during the life cycle of Cryptosporidium parvum grown in vitro These different classes of inhibitors targeted different stages of the life cycle, including compounds that blocked replication (PheRS inhibitors), prevented the segmentation of daughter cells and thus blocked egress (PI4K inhibitors), or affected sexual-stage development (a piperazine compound of unknown mechanism). Long-term cultivation of C. parvum in epithelial cell monolayers derived from intestinal stem cells was used to distinguish between cidal and static activities based on the ability of parasites to recover from treatment. Collectively, these approaches should aid in identifying mechanisms of action and for designing in vivo efficacy studies based on time-dependent concentrations needed to achieve cidal activity. IMPORTANCE Currently, nitazoxanide is the only FDA-approved treatment for cryptosporidiosis; unfortunately, it is ineffective in immunocompromised patients, has varied efficacy in immunocompetent individuals, and is not approved in infants under 1 year of age. Identifying new inhibitors for the treatment of cryptosporidiosis requires standardized and quantifiable in vitro assays for assessing potency, selectivity, timing of activity, and reversibility. Here, we provide new protocols for defining which stages of the life cycle are susceptible to four highly active compound classes that likely inhibit different targets in the parasite. We also utilize a newly developed long-term culture system to define assays for monitoring reversibility as a means of defining cidal activity as a function of concentration and time of treatment. These assays should provide valuable in vitro parameters to establish conditions for efficacious in vivo treatment., (Copyright © 2020 Funkhouser-Jones et al.)

Toxoplasma is a widespread parasite of animals including many rodents that are a natural part of the transmission cycle between cats, which serve as the definitive host. Although wild rodents, including house mice, are relatively resistant, laboratory mice are highly susceptible to infection. As such, laboratory mice have been used to compare pathogenesis of natural variants and to evaluate the contributions of both host and parasite genes to infection. Protocols are provided here for evaluating acute and chronic infection with different parasite strains in laboratory mice. These protocols should provide uniform standards for evaluating natural variants and attenuated mutants and for comparing outcomes across different studies and between different laboratories.

Micronemes are specialized secretory organelles present in all motile forms of apicomplexan parasites. Microneme vesicles hold adhesins and other proteins that are secreted to facilitate parasite attachment, invasion of host cells, and egress following replication-all processes indispensable for cell-to-cell transmission of these obligate intracellular parasites. Defining the signaling pathways that lead to microneme secretion is an important part of understanding the infectious cycle of apicomplexan parasites. However, the classical method of measuring microneme secretion by immunoblotting for microneme proteins in parasite excreted/secreted antigen (ESA) preparations is low-throughput and only semiquantitative. We recently reported a new luciferase-based method for measuring microneme secretion in a 96-well format with high sensitivity in the model apicomplexan Toxoplasma gondii. Here, we aim to elaborate on this detection method and review current practices for stimulating microneme secretion in vitro.

Cryptosporidium parvum has a complex life cycle consisting of asexual and sexual phases that culminate in oocyst formation in vivo. The most widely used cell culture platforms to study C. parvum only support a few days of growth and do not allow the parasite to proceed past the sexual stages to complete oocyst formation. Additionally, these cell culture platforms are mostly adenocarcinoma cell lines, which do not adequately model the parasite's natural environment in the small intestine. We describe here a method to create primary intestinal epithelial cell monolayers that support long-term C. parvum growth. Monolayers were derived from mouse intestinal stem cells grown as spheroids and plated onto transwells, allowing for separate apical and basolateral compartments. In the apical chamber, the cell growth medium was removed to create an "air-liquid interface" that enhanced host cell differentiation and supported long-term C. parvum growth. The use of primary intestinal cells to grow C. parvum in vitro will be a valuable tool for studying host-parasite interactions using a convenient in vitro model that more closely resembles the natural niche in the intestine.

Artemisinins are effective against a variety of parasites and provide the first line of treatment for malaria. Laboratory studies have identified several mechanisms for artemisinin resistance in Plasmodium falciparum , including mutations in Kelch13 that are associated with delayed clearance in some clinical isolates, although other mechanisms are likely involved. To explore other potential mechanisms of resistance in parasites, we took advantage of the genetic tractability of Toxoplasma gondii , a related parasite that shows moderate sensitivity to artemisinin. Resistant populations of T. gondii were selected by culture in increasing concentrations and whole-genome sequencing identified several nonconservative point mutations that emerged in the population and were fixed over time. Genome editing using CRISPR/Cas9 was used to introduce point mutations conferring amino acid changes in a serine protease homologous to DegP and a serine/threonine protein kinase of unknown function. Single and double mutations conferred a competitive advantage over wild-type parasites in the presence of drug, despite not changing EC 50 values. Additionally, the evolved resistant lines showed dramatic amplification of the mitochondria genome, including genes encoding cytochrome b and cytochrome c oxidase I. Prior studies in yeast and mammalian tumor cells implicate the mitochondrion as a target of artemisinins, and treatment of wild-type parasites with high concentrations of drug decreased mitochondrial membrane potential, a phenotype that was stably altered in the resistant parasites. These findings extend the repertoire of mutations associated with artemisinin resistance and suggest that the mitochondrion may be an important target of inhibition of resistance in T. gondii .

The colonic epithelium can undergo multiple rounds of damage and repair, often in response to excessive inflammation. The responsive stem cell that mediates this process is unclear, in part because of a lack of in vitro models that recapitulate key epithelial changes that occur in vivo during damage and repair. Here, we identify a Hopx + colitis-associated regenerative stem cell (CARSC) population that functionally contributes to mucosal repair in mouse models of colitis. Hopx + CARSCs, enriched for fetal-like markers, transiently arose from hypertrophic crypts known to facilitate regeneration. Importantly, we established a long-term, self-organizing two-dimensional (2D) epithelial monolayer system to model the regenerative properties and responses of Hopx + CARSCs. This system can reenact the "homeostasis-injury-regeneration" cycles of epithelial alterations that occur in vivo. Using this system, we found that hypoxia and endoplasmic reticulum stress, insults commonly present in inflammatory bowel diseases, mediated the cyclic switch of cellular status in this process., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The protozoan parasite Toxoplasma gondii is thought to exploit monocyte trafficking to facilitate dissemination across endothelial barriers such as the blood-brain barrier. Here, we analysed the migration of parasitized monocytes in model endothelial and interstitial environments. We report that infection enhanced monocyte locomotion on the surface of endothelial cells, but profoundly inhibited monocyte transmigration across endothelial barriers. By contrast, infection robustly increased monocyte and macrophage migration through collagen-rich tissues in a Rho-ROCK-dependent manner consistent with integrin-independent interstitial migration. We further demonstrated that the secreted T. gondii protein kinase ROP17 was required for enhanced tissue migration. In vivo, ROP17-deficient parasites failed to upregulate monocyte tissue migration and exhibited an early dissemination delay, leading to prolonged mouse survival. Our findings indicate that the parasite-induced changes in monocyte motility primarily facilitate the transport of T. gondii through tissues and promote systemic dissemination, rather than shuttle parasites across the blood-brain barrier via extravasation.

Toxoplasma gondii (T. gondii) is a parasitic protist that can infect nearly all nucleated cell types and tissues of warm-blooded vertebrate hosts. T. gondii utilises a unique form of gliding motility to cross cellular barriers, enter tissues, and penetrate host cells, thus enhancing spread within an infected host. However, T. gondii also disseminates by hijacking the migratory abilities of infected leukocytes. Traditionally, this process has been viewed as a route to cross biological barriers such as the blood-brain barrier. Here, we review recent findings that challenge this view by showing that infection of monocytes downregulates the program of transendothelial migration. Instead, infection by T. gondii enhances Rho-dependent interstitial migration of monocytes and macrophages, which enhances dissemination within tissues. Collectively, the available evidence indicates that T. gondii parasites use multiple means to disseminate within the host, including enhanced motility in tissues and translocation across biological barriers., (© 2019 John Wiley & Sons Ltd.)

Antimicrobial treatment failure threatens our ability to control infections. In addition to antimicrobial resistance, treatment failures are increasingly understood to derive from cells that survive drug treatment without selection of genetically heritable mutations. Parasitic protozoa, such as Plasmodium species that cause malaria, Toxoplasma gondii and kinetoplastid protozoa, including Trypanosoma cruzi and Leishmania spp., cause millions of deaths globally. These organisms can evolve drug resistance and they also exhibit phenotypic diversity, including the formation of quiescent or dormant forms that contribute to the establishment of long-term infections that are refractory to drug treatment, which we refer to as 'persister-like cells'. In this Review, we discuss protozoan persister-like cells that have been linked to persistent infections and discuss their impact on therapeutic outcomes following drug treatment.

In contrast to the importance of type II interferon-γ (IFN-γ) in control of toxoplasmosis, the role of type I IFN is less clear. We demonstrate here that TgIST, a secreted effector previously implicated in blocking type II IFN-γ signaling, also blocked IFN-β responses by inhibiting STAT1/STAT2-mediated transcription in infected cells. Consistent with a role for type I IFN in cell intrinsic control, ∆ Tgis t mutants were more susceptible to growth inhibition by murine and human macrophages activated with IFN-β. Additionally, type I IFN was important for production of IFN-γ by natural killer (NK) cells and recruitment of inflammatory monocytes at the site of infection. Mice lacking type I IFN receptors (Ifnar1 -/- ) showed increased mortality following infection with wild-type parasites and decreased virulence of ∆Tgist parasites was restored in Ifnar1 -/- mice. The findings highlight the importance of type I IFN in control of toxoplasmosis and illuminate a parasite mechanism to counteract the effects of both type I and II IFN-mediated host defenses., Competing Interests: The authors declare no conflict of interest.

Innate lymphoid cells (ILCs) were originally classified based on their cytokine profiles, placing natural killer (NK) cells and ILC1s together, but recent studies support their separation into different lineages at steady-state. However, tumors may induce NK cell conversion into ILC1-like cells that are limited to the tumor microenvironment and whether this conversion occurs beyond this environment remains unknown. Here, we describe Toxoplasma gondii infection converts NK cells into ILC1-like cells that are distinct from both steady-state NK cells and ILC1s in uninfected mice. These cells were Eomes-dependent, indicating that NK cells can give rise to Eomes - Tbet-dependent ILC1-like cells that circulate widely and persist independent of ongoing infection. Moreover, these changes appear permanent, as supported by epigenetic analyses. Thus, these studies markedly expand current concepts of NK cells, ILCs, and their potential conversion., Competing Interests: EP, SP, QW, PA, KZ, SP, MH, MB, BP, PC, MC, KM, EO, MA, LS, WY No competing interests declared, (© 2019, Park et al.)

Despite being a frequent cause of severe diarrheal disease in infants and an opportunistic infection in immunocompromised patients, Cryptosporidium research has lagged due to a lack of facile experimental methods. Here, we describe a platform for complete life cycle development and long-term growth of C. parvum in vitro using "air-liquid interface" (ALI) cultures derived from intestinal epithelial stem cells. Transcriptomic profiling revealed that differentiating epithelial cells grown under ALI conditions undergo profound changes in metabolism and development that enable completion of the parasite life cycle in vitro. ALI cultures support parasite expansion > 100-fold and generate viable oocysts that are transmissible in vitro and to mice, causing infection and animal death. Transgenic parasite lines created using CRISPR/Cas9 were used to complete a genetic cross in vitro, demonstrating Mendelian segregation of chromosomes during meiosis. ALI culture provides an accessible model that will enable innovative studies into Cryptosporidium biology and host interactions., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The gastrointestinal tract hosts the largest collection of commensal microbes in the body. Infections at this site can cause significant perturbations in the microbiota, known as dysbiosis, that facilitate the expansion of pathobionts, and can elicit inappropriate immune responses that impair the intestinal barrier function. Dysbiosis typically occurs during intestinal infection with Toxoplasma gondii . Host resistance to T. gondii depends on a potent Th1 response. In addition, a Th17 response is also elicited. How Th17 cells contribute to the host response to T. gondii remains unclear. Here we show that class I-restricted T cell-associated molecule (CRTAM) expression on T cells is required for an optimal IL-17 production during T. gondii infection. Moreover, that the lack of IL-17, results in increased immunopathology caused by an impaired antimicrobial peptide production and bacterial translocation from the intestinal lumen to the mesenteric lymph nodes and spleen.

Microneme (MIC) proteins play important roles in the recognition, adhesion, and invasion of host cells by Toxoplasma gondii Previous studies have shown that MIC proteins are highly immunogenic in the mouse and recognized by human serum antibodies. Here we report that T. gondii antigens MIC1, MIC3, MIC4, and MIC6 were capable of inducing memory responses leading to production of gamma interferon (IFN-γ) by T cells from T. gondii -infected mice. Production of IFN-γ was demonstrated using enzyme-linked immunosorbent spot (ELISPOT) assay and also intracellular cytokine staining. All four MIC antigens displayed very high sensitivity (100%) and specificity (86 to 100%) for detecting chronic infection. Interestingly, IFN-γ was produced by both CD4 + and CD8 + T cells in BALB/c mice but primarily by CD4 + T cells in C57BL/6 mice. Phenotypic characterization of IFN-γ-producing CD4 + and CD8 + T cells in BALB/c mice and CD4 + T cells in C57BL/6 mice revealed effector memory T cells (CD44 hi CD62L lo ) as the predominant cells that contributed to IFN-γ production in response to MIC antigens. Effector memory responses were seen in mice of different major histocompatibility complex class II (MHC-II) haplotypes, suggesting that MIC antigens contain epitopes that are broadly recognized. IMPORTANCE Current diagnosis of toxoplasmosis relies almost exclusively on antibody detection, and while detection of IgG provides a useful estimate of prior infection, it does not alone indicate immune status. In contrast, detection of IFN-γ responses to T. gondii antigens has been used to monitor immune responsiveness in HIV-infected patients, thus providing valuable predictions about the potential for disease reactivation. However, specific T. gondii antigens that can be used in assays to detect cellular immunity remain largely undefined. In this study, we examined the diagnostic potential of microneme antigens of T. gondii using IFN-γ detection assays. Our findings demonstrate that MIC antigens (MIC1, MIC3, MIC4, and MIC6) elicit IFN-γ responses from memory T cells in chronically infected mice. Monitoring IFN-γ production by T cells stimulated with MIC antigens provided high sensitivity and specificity for detection of T. gondii infection in mice. Taken together, these studies suggest that microneme antigens might be useful as an adjunct to serological testing to monitor immune status during infection., (Copyright © 2019 Saraav et al.)

A safer treatment for toxoplasmosis would be achieved by improving the selectivity and potency of dihydrofolate reductase (DHFR) inhibitors, such as pyrimethamine (1), for Toxoplasma gondii DHFR ( TgDHFR) relative to human DHFR ( hDHFR). We previously reported on the identification of meta-biphenyl analog 2, designed by in silico modeling of key differences in the binding pocket between TgDHFR and hDHFR. Compound 2 improves TgDHFR selectivity 6.6-fold and potency 16-fold relative to 1. Here, we report on the optimization and structure-activity relationships of this arylpiperazine series leading to the discovery of 5-(4-(3-(2-methoxypyrimidin-5-yl)phenyl)piperazin-1-yl)pyrimidine-2,4-diamine 3. Compound 3 has a TgDHFR IC 50 of 1.57 ± 0.11 nM and a hDHFR to TgDHFR selectivity ratio of 196, making it 89-fold more potent and 16-fold more selective than 1. Compound 3 was highly effective in control of acute infection by highly virulent strains of T. gondii in the murine model, and it possesses the best combination of selectivity, potency, and prerequisite drug-like properties to advance into IND-enabling, preclinical development.

Apicomplexan parasites rely on cyclic nucleotide-dependent kinases for host cell infection, yet the mechanisms that control their activation remain unknown. Here we show that an apically localized guanylate cyclase (GC) controls microneme secretion and lytic growth in the model apicomplexan Toxoplasma gondii. Cell-permeable cGMP reversed the block in microneme secretion seen in a knockdown of TgGC, linking its function to production of cGMP. TgGC possesses an N-terminal P-type ATPase domain fused to a C-terminal heterodimeric guanylate cyclase domain, an architecture found only in Apicomplexa and related protists. Complementation with a panel of mutants revealed a critical requirement for the P-type ATPase domain for maximum GC function. We further demonstrate that knockdown of TgGC in vivo protects mice from lethal infection by blocking parasite expansion and dissemination. Collectively, this work demonstrates that cGMP-mediated signaling in Toxoplasma relies on a multi-domain architecture, which may serve a conserved role in related parasites., (Copyright © 2018 Elsevier Inc. All rights reserved.)

During the process of cross-presentation, viral or tumor-derived antigens are presented to CD8 + T cells by Batf3- dependent CD8α + /XCR1 + classical dendritic cells (cDC1s). We designed a functional CRISPR screen for previously unknown regulators of cross-presentation, and identified the BEACH domain-containing protein WDFY4 as essential for cross-presentation of cell-associated antigens by cDC1s in mice. However, WDFY4 was not required for major histocompatibility complex class II presentation, nor for cross-presentation by monocyte-derived dendritic cells. In contrast to Batf3 -/- mice, Wdfy4 -/- mice displayed normal lymphoid and nonlymphoid cDC1 populations that produce interleukin-12 and protect against Toxoplasma gondii infection. However, similar to Batf3 -/- mice, Wdfy4 -/- mice failed to prime virus-specific CD8 + T cells in vivo or induce tumor rejection, revealing a critical role for cross-presentation in antiviral and antitumor immunity., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Toxoplasma gondii is an obligate intracellular parasite capable of causing severe disease due to congenital infection and in patients with compromised immune systems. Control of infection is dependent on a robust Th1 type immune response including production of interferon gamma (IFN-γ), which is essential for control. IFN-γ activates a variety of antimicrobial mechanisms in host cells, which are then able to control intracellular parasites such as T. gondii. Despite the effectiveness of these pathways in controlling acute infection, the immune system is unable to eradicate chronic infections that can persist for life. Similarly, while antibiotic treatment can control acute infection, it is unable to eliminate chronic infection. To identify compounds that would act synergistically with IFN-γ, we performed a high-throughput screen of diverse small molecule libraries to identify inhibitors of T. gondii. We identified a number of compounds that inhibited parasite growth in vitro at low μM concentrations and that demonstrated enhanced potency in the presence of a low level of IFN-γ. A subset of these compounds act by enhancing the recruitment of light chain 3 (LC3) to the parasite-containing vacuole, suggesting they work by an autophagy-related process, while others were independent of this pathway. The pattern of IFN-γ dependence was shared among the majority of analogs from 6 priority scaffolds, and analysis of structure activity relationships for one such class revealed specific stereochemistry associated with this feature. Identification of these IFN-γ-dependent leads may lead to development of improved therapeutics due to their synergistic interactions with immune responses.

Phagocytic cells are the first line of innate defense against intracellular pathogens, and yet Toxoplasma gondii is renowned for its ability to survive in macrophages, although this paradigm is based on virulent type I parasites. Surprisingly, we find that avirulent type III parasites are preferentially cleared in naive macrophages, independent of gamma interferon (IFN-γ) activation. The ability of naive macrophages to clear type III parasites was dependent on enhanced activity of NADPH oxidase (Nox)-generated reactive oxygen species (ROS) and induction of guanylate binding protein 5 (Gbp5). Macrophages infected with type III parasites (CTG strain) showed a time-dependent increase in intracellular ROS generation that was higher than that induced by type I parasites (GT1 strain). The absence of Nox1 or Nox2, gp91 subunit isoforms of the Nox complex, reversed ROS-mediated clearance of CTG parasites. Consistent with this finding, both Nox1 -/- and Nox2 -/- mice showed higher susceptibility to CTG infection than wild-type mice. Additionally, Gbp5 expression was induced upon infection and the enhanced clearance of CTG strain parasites was reversed in Gbp5 -/- macrophages. Expression of a type I ROP18 allele in CTG prevented clearance in naive macrophages, suggesting that it plays a role counteracting Gbp5. Although ROS and Gbp5 have been linked to activation of the NLRP3 inflammasome, clearance of CTG parasites did not rely on induction of pyroptosis. Collectively, these findings reveal that not all strains of T. gondii are adept at avoiding clearance in macrophages and define new roles for ROS and Gbps in controlling this important intracellular pathogen. IMPORTANCE Toxoplasma infections in humans and other mammals are largely controlled by IFN-γ produced by the activated adaptive immune system. However, we still do not completely understand the role of cell-intrinsic functions in controlling Toxoplasma or other apicomplexan infections. The present work identifies intrinsic activities in naive macrophages in counteracting T. gondii infection. Using an avirulent strain of T. gondii , we highlight the importance of Nox complexes in conferring protection against parasite infection both in vitro and in vivo We also identify Gbp5 as a novel macrophage factor involved in limiting intracellular infection by avirulent strains of T. gondii The rarity of human infections caused by type III strains suggests that these mechanisms may also be important in controlling human toxoplasmosis. These findings further extend our understanding of host responses and defense mechanisms that act to control parasitic infections at the cellular level., (Copyright © 2018 Matta et al.)

Toxoplasma gondii , the causative agent of toxoplasmosis, is a widespread intracellular parasite able to infect virtually any nucleated cell. T. gondii infection of activated macrophages inhibits nitric oxide (NO) production; however, parasite effectors responsible for this block have not been defined. Macrophage populations are extremely heterogeneous, responding differently to stimuli and to parasite infection. Here we evaluated the inhibition of NO production caused by T. gondii infection of J774-A1 and RAW 264.7 macrophages and assessed the role of several known parasite virulence factors in this phenotype. Infection of activated macrophages from both macrophage lines reduced NO production, however, the mechanism of this decrease was different. Consistent with previous reports, infected J774-A1 macrophages had reduced iNOS expression and lower number of iNOS positive cells. In contrast, T. gondii infection of RAW 264.7 macrophages did not alter iNOS expression or the number of iNOS positive cells, and yet it led to lower levels of NO production. Deletion of a number of previously defined virulence factors including ROP kinases that disrupt innate immune factors, TgIST which blocks STAT1 activation, as well as the secretory trafficking proteins ASP5 and MYR1, did not alter the phenotype of decreased NO production. Taken together our findings indicate that T. gondii infection inhibits NO production of activated macrophages by different mechanisms that involve reduction of iNOS expression vs. iNOS impairment, and suggest that a novel parasite effector is involved in modulating this important host defense pathway.

Toxoplasma gondii is a common zoonotic infection of humans, and estimates indicate that 1-2 billion people are chronically infected. Although largely asymptomatic, chronic infection poses risk of serious disease due to reactivation should immunity decline. Current therapies for toxoplasmosis only control acute infection caused by actively proliferating tachyzoites but do not eradicate the chronic tissue cyst stages. As well, there are considerable adverse side effects of the most commonly used therapy of combined sulfadiazine and pyrimethamine. Targeting the folate pathway is also an effective treatment for malaria, caused by the related parasites Plasmodium spp., suggesting common agents might be used to treat both infections. Here, we evaluated currently approved and newly emerging medicines for malaria to determine if such compounds might also prove useful for treating toxoplasmosis. Surprisingly, the majority of antimalarial compounds being used currently or in development for treatment of malaria were only modestly effective at inhibiting in vitro growth of T. gondii tachyzoites. These findings suggest that many essential processes in P. falciparum that are targeted by antimalarial compounds are either divergent or nonessential in T. gondii, thus limiting options for repurposing of current antimalarial medicines for toxoplasmosis.

Infection with the protozoan parasite, Toxoplasma gondii (T. gondii), has been associated with the increased risk for several psychiatric disorders. The exact mechanisms of a hypothesized contribution of T. gondii infection are poorly understood. The T. gondii genome contains two aromatic amino acid hydroxylase genes (AAH1 and AAH2) that encode proteins that can produce L-DOPA. One popular hypothesis posits that these encoded enzymes might influence dopamine (DA) production and hence DA synaptic transmission, leading to neurobehavioral abnormalities in the infected host. Prior studies have shown that deletion of these genes does not alter DA levels in the brain or exploratory activity in infected mice. However, possible effects of AAH gene deficiency on infection-induced brain and behavior alterations that are directly linked to DA synaptic transmission have not been evaluated. We found that chronic T. gondii infection of BALB/c mice leads to blunted response to amphetamine or cocaine and decreased expression of Dopamine Transporter (DAT) and Vesicular Monoamine Transporter 2 (VMAT2). Deletion of AAH2 had no effects on these changes in infected mice. Both wild type and Δaah2 strains produced comparable levels of neuroinflammation. Our findings demonstrate that AAH2 is not required for T. gondii infection-produced DA-dependent neurobehavioral abnormalities., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Among the obstacles hindering Cryptosporidium research is the lack of an in vitro culture system that supports complete life development and propagation. This major barrier has led to a shortage of widely available anti- Cryptosporidium antibodies and a lack of markers for staging developmental progression. Previously developed antibodies against Cryptosporidium were raised against extracellular stages or recombinant proteins, leading to antibodies with limited reactivity across the parasite life cycle. Here we sought to create antibodies that recognize novel epitopes that could be used to define intracellular development. We identified a mouse epithelial cell line that supported C. parvum growth, enabling immunization of mice with infected cells to create a bank of monoclonal antibodies (MAbs) against intracellular parasite stages while avoiding the development of host-specific antibodies. From this bank, we identified 12 antibodies with a range of reactivities across the parasite life cycle. Importantly, we identified specific MAbs that can distinguish different life cycle stages, such as trophozoites, merozoites, type I versus II meronts, and macrogamonts. These MAbs provide valuable tools for the Cryptosporidium research community and will facilitate future investigation into parasite biology. IMPORTANCE Cryptosporidium is a protozoan parasite that causes gastrointestinal disease in humans and animals. Currently, there is a limited array of antibodies available against the parasite, which hinders imaging studies and makes it difficult to visualize the parasite life cycle in different culture systems. In order to alleviate this reagent gap, we created a library of novel antibodies against the intracellular life cycle stages of Cryptosporidium We identified antibodies that recognize specific life cycle stages in distinctive ways, enabling unambiguous description of the parasite life cycle. These MAbs will aid future investigation into Cryptosporidium biology and help illuminate growth differences between various culture platforms., (Copyright © 2018 Wilke et al.)

Defining protein interaction networks can provide key insights into how protein complexes govern complex biological problems. Here we define a method for proximity based labeling using permissive biotin ligase to define protein networks in the intracellular parasite Toxoplasma gondii . When combined with CRISPR/Cas9 based tagging, this method provides a robust approach to defining protein networks. This approach detects interaction within intact cells, it is applicable to both soluble and insoluble components, including large proteins complexes that interact with the cytoskeleton and unique microtubule organizing center that comprises the apical complex in apicomplexan parasites., Competing Interests: The authors declare that there are no conflicts or competing interests.

Toxoplasma gondii is a member of the deadly phylum of protozoan parasites called Apicomplexa. As a model apicomplexan, there is a great wealth of information regarding T. gondii 's 8,000+ protein coding genes including sequence variation, expression, and relative contribution to parasite fitness. However, new tools are needed to functionally investigate hundreds of putative essential protein coding genes. Accordingly, we recently implemented the auxin-inducible degron (AID) system for studying essential proteins in T. gondii . Here we provide a step-by-step protocol for examining protein function in T. gondii using the AID system in a tissue culture setting.

Calcium dependent protein kinase 1 (CDPK1) is an essential enzyme in the opportunistic pathogen Toxoplasma gondii. CDPK1 controls multiple processes that are critical to the intracellular replicative cycle of T. gondii including secretion of adhesins, motility, invasion, and egress. Remarkably, CDPK1 contains a small glycine gatekeeper residue in the ATP binding pocket making it sensitive to ATP-competitive inhibitors with bulky substituents that complement this expanded binding pocket. Here we explored structure-activity relationships of a series of pyrazolopyrimidine inhibitors of CDPK1 with the goal of increasing selectivity over host enzymes, improving antiparasite potency, and improving metabolic stability. The resulting lead compound 24 exhibited excellent enzyme inhibition and selectivity for CDPK1 and potently inhibited parasite growth in vitro. Compound 24 was also effective at treating acute toxoplasmosis in the mouse, reducing dissemination to the central nervous system, and decreasing reactivation of chronic infection in severely immunocompromised mice. These findings provide proof of concept for the development of small molecule inhibitors of CDPK1 for treatment of CNS toxoplasmosis.

Apicomplexan parasites are typified by an apical complex that contains a unique microtubule-organizing center (MTOC) that organizes the cytoskeleton. In apicomplexan parasites such as Toxoplasma gondii, the apical complex includes a spiral cap of tubulin-rich fibers called the conoid. Although described ultrastructurally, the composition and functions of the conoid are largely unknown. Here, we localize 11 previously undescribed apical proteins in T. gondii and identify an essential component named conoid protein hub 1 (CPH1), which is conserved in apicomplexan parasites. CPH1 contains ankyrin repeats that are required for structural integrity of the conoid, parasite motility, and host cell invasion. Proximity labeling and protein interaction network analysis reveal that CPH1 functions as a hub linking key motor and structural proteins that contain intrinsically disordered regions and coiled coil domains. Our findings highlight the importance of essential protein hubs in controlling biological networks of MTOCs in early-branching protozoan parasites.

Early electron microscopy studies revealed the elaborate cellular features that define the unique adaptations of apicomplexan parasites. Among these were bulbous rhoptry (ROP) organelles and small, dense granules (GRAs), both of which are secreted during invasion of host cells. These early morphological studies were followed by the exploration of the cellular contents of these secretory organelles, revealing them to be comprised of highly divergent protein families with few conserved domains or predicted functions. In parallel, studies on host-pathogen interactions identified many host signaling pathways that were mysteriously altered by infection. It was only with the advent of forward and reverse genetic strategies that the connections between individual parasite effectors and the specific host pathways that they targeted finally became clear. The current repertoire of parasite effectors includes ROP kinases and pseudokinases that are secreted during invasion and that block host immune pathways. Similarly, many secretory GRA proteins alter host gene expression by activating host transcription factors, through modification of chromatin, or by inducing small noncoding RNAs. These effectors highlight novel mechanisms by which T. gondii has learned to harness host signaling to favor intracellular survival and will guide future studies designed to uncover the additional complexity of this intricate host-pathogen interaction., (Copyright © 2017 American Society for Microbiology.)

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion.

Cyclic GMP (cGMP)-dependent protein kinase (protein kinase G [PKG]) is essential for microneme secretion, motility, invasion, and egress in apicomplexan parasites, However, the separate roles of two isoforms of the kinase that are expressed by some apicomplexans remain uncertain. Despite having identical regulatory and catalytic domains, PKG I is plasma membrane associated whereas PKG II is cytosolic in Toxoplasma gondii To determine whether these isoforms are functionally distinct or redundant, we developed an auxin-inducible degron (AID) tagging system for conditional protein depletion in T. gondii By combining AID regulation with genome editing strategies, we determined that PKG I is necessary and fully sufficient for PKG-dependent cellular processes. Conversely, PKG II is functionally insufficient and dispensable in the presence of PKG I The difference in functionality mapped to the first 15 residues of PKG I , containing a myristoylated Gly residue at position 2 that is critical for membrane association and PKG function. Collectively, we have identified a novel requirement for cGMP signaling at the plasma membrane and developed a new system for examining essential proteins in T. gondii IMPORTANCE Toxoplasma gondii is an obligate intracellular apicomplexan parasite and important clinical and veterinary pathogen that causes toxoplasmosis. Since apicomplexans can only propagate within host cells, efficient invasion is critically important for their life cycles. Previous studies using chemical genetics demonstrated that cyclic GMP signaling through protein kinase G (PKG)-controlled invasion by apicomplexan parasites. However, these studies did not resolve functional differences between two compartmentalized isoforms of the kinase. Here we developed a conditional protein regulation tool to interrogate PKG isoforms in T. gondii We found that the cytosolic PKG isoform was largely insufficient and dispensable. In contrast, the plasma membrane-associated isoform was necessary and fully sufficient for PKG function. Our studies identify the plasma membrane as a key location for PKG activity and provide a broadly applicable system for examining essential proteins in T. gondii ., (Copyright © 2017 Brown et al.)

The Toxoplasma gondii genome contains two aromatic amino acid hydroxylase genes, AAH1 and AAH2 encode proteins that produce L-DOPA, which can serve as a precursor of catecholamine neurotransmitters. It has been suggested that this pathway elevates host dopamine levels thus making infected rodents less fearful of their definitive Felidae hosts. However, L-DOPA is also a structural precursor of melanins, secondary quinones, and dityrosine protein crosslinks, which are produced by many species. For example, dityrosine crosslinks are abundant in the oocyst walls of Eimeria and T. gondii, although their structural role has not been demonstrated, Here, we investigated the biology of AAH knockout parasites in the sexual reproductive cycle within cats. We found that ablation of the AAH genes resulted in reduced infection in the cat, lower oocyst yields, and decreased rates of sporulation. Our findings suggest that the AAH genes play a predominant role during infection in the gut of the definitive feline host.

Toxoplasma gondii is an apicomplexan parasite that secretes a large number of protein kinases and pseudokinases from its rhoptry organelles. Although some rhoptry kinases (ROPKs) act as virulence factors, many remain uncharacterized. In this study, predicted ROPKs were assessed for bradyzoite expression then prioritized for a reverse genetic analysis in the type II strain Pru that is amenable to targeted disruption. Using CRISPR/Cas9, we engineered C-terminally epitope tagged ROP21 and ROP27 and demonstrated their localization to the parasitophorous vacuole and cyst matrix. ROP21 and ROP27 were not secreted from microneme, rhoptry, or dense granule organelles, but rather were located in small vesicles consistent with a constitutive pathway. Using CRISPR/Cas9, the genes for ROP21, ROP27, ROP28, and ROP30 were deleted individually and in combination, and the mutant parasites were assessed for growth and their ability to form tissue cysts in mice. All knockouts lines were normal for in vitro growth and bradyzoite differentiation, but a combined ∆rop21/∆rop17 knockout led to a 50% reduction in cyst burden in vivo. Our findings question the existing annotation of ROPKs based solely on bioinformatic techniques and yet highlight the importance of secreted kinases in determining the severity of chronic toxoplasmosis., (© 2016 John Wiley & Sons Ltd.)

Efficient and site-specific alteration of the genome is key to decoding and altering the genomic information of an organism. Over the last couple of years, the RNA-guided Cas9 nucleases derived from the prokaryotic type 2 CRISPR (clustered regularly interspaced short palindromic repeats) systems have drastically improved our ability to engineer the genomes of a variety of organisms including Toxoplasma gondii. In this chapter, we describe detailed protocols for using the CRISPR/Cas9 system adapted from Streptococcus pyogenes to perform efficient genetic manipulations in T. gondii such as gene disruption, gene tagging and genetic complementation. The technical details of the strategy, including CRISPR plasmid construction, target construct generation, parasite transfection and positive clone identification are also provided. These methods are easy to customize to any gene of interest (GOI) and will greatly accelerate studies on this important pathogen.

Background: Phosphoinositides (PIs) and their derivatives are essential cellular components that form the building blocks for cell membranes and regulate numerous cell functions. Specifically, the ability to generate myo-inositol 1,4,5-trisphosphate (InsP3) via phospholipase C (PLC) dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to InsP3 and diacylglycerol (DAG) initiates intracellular calcium signaling events representing a fundamental signaling mechanism dependent on PIs. InsP3 produced by PI turnover as a second messenger causes intracellular calcium release, especially from endoplasmic reticulum, by binding to the InsP3 receptor (InsP3R). Various PIs and the enzymes, such as phosphatidylinositol synthase and phosphatidylinositol 4-kinase, necessary for their turnover have been characterized in Apicomplexa, a large phylum of mostly commensal organisms that also includes several clinically relevant parasites. However, InsP3Rs have not been identified in genomes of apicomplexans, despite evidence that these parasites produce InsP3 that mediates intracellular Ca2+ signaling., Conclusion: Evidence to supporting IP3-dependent signaling cascades in apicomplexans suggests that they may harbor a primitive or non-canonical InsP3R. Understanding these pathways may be informative about early branching eukaryotes, where such signaling pathways also diverge from animal systems, thus identifying potential novel and essential targets for therapeutic intervention., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Apicomplexan parasites contain a conserved protein CelTOS that, in malaria parasites, is essential for traversal of cells within the mammalian host and arthropod vector. However, the molecular role of CelTOS is unknown because it lacks sequence similarity to proteins of known function. Here, we determined the crystal structure of CelTOS and discovered CelTOS resembles proteins that bind to and disrupt membranes. In contrast to known membrane disruptors, CelTOS has a distinct architecture, specifically binds phosphatidic acid commonly present within the inner leaflet of plasma membranes, and potently disrupts liposomes composed of phosphatidic acid by forming pores. Microinjection of CelTOS into cells resulted in observable membrane damage. Therefore, CelTOS is unique as it achieves nearly universal inner leaflet cellular activity to enable the exit of parasites from cells during traversal. By providing novel molecular insight into cell traversal by apicomplexan parasites, our work facilitates the design of therapeutics against global pathogens., Competing Interests: The authors declare that no competing interests exist.

Toxoplasma gondii is a widespread parasite of warm-blooded vertebrates that also causes opportunistic infections in humans. Rodents are a natural host for asexually replicating forms, whereas cats serve as the definitive host for sexual development. The laboratory mouse provides a model to study pathogenesis. Strains of T. gondii are globally diverse, with more than 16 distinct haplogroups clustered into 6 major clades. Forward genetic analysis of genetic crosses between different lineages has been used to define the molecular basis of acute virulence in the mouse. These studies have identified a family of secretory serine/threonine rhoptry kinases that target innate immune pathways to protect intracellular parasites from destruction. Rhoptry kinases target immunity-related GTPases, a family of immune effectors that is expanded in rodents. Similar forward genetic studies may be useful to define the basis of pathogenesis in other hosts, including humans, where infections of different strains present with variable clinical severity.

Interferon gamma (IFN-γ) is an essential mediator of host defense against intracellular pathogens, including the protozoan parasite Toxoplasma gondii. However, prior T. gondii infection blocks IFN-γ-dependent gene transcription, despite the downstream transcriptional activator STAT1 being activated and bound to cognate nuclear promoters. We identify the parasite effector that blocks STAT1-dependent transcription and show it is associated with recruitment of the Mi-2 nucleosome remodeling and deacetylase (NuRD) complex, a chromatin-modifying repressor. This secreted effector, toxoplasma inhibitor of STAT1-dependent transcription (TgIST), translocates to the host cell nucleus, where it recruits Mi-2/NuRD to STAT1-dependent promoters, resulting in altered chromatin and blocked transcription. TgIST is conserved across strains, underlying their shared ability to block IFN-γ-dependent transcription. TgIST deletion results in increased parasite clearance in IFN-γ-activated cells and reduced mouse virulence, which is restored in IFN-γ-receptor-deficient mice. These findings demonstrate the importance of both IFN-γ responses and the ability of pathogens to counteract these defenses., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Bioluminescent reporter assays have been widely used to study the effect of Toxoplasma gondii on host gene expression. In the present study, we extend these studies by engineering novel reporter cell lines containing a gamma-activated sequence (GAS) element driving firefly luciferase (FLUC). In RAW264.7 macrophages, T. gondii type I strain (GT1) infection blocked interferon gamma (IFN-γ)-induced FLUC activity to a significantly greater extent than infection by type II (ME49) and type III (CTG) strains. Quantitative trait locus (QTL) analysis of progeny from a prior genetic cross identified a genomic region on chromosome XII that correlated with the observed strain-dependent phenotype. This QTL region contains two isoforms of the T. gondii enzyme nucleoside triphosphate hydrolase (NTPase) that were the prime candidates for mediating the observed strain-specific effect. Using reverse genetic analysis we show that deletion of NTPase I from a type I strain (RH) background restored the higher luciferase levels seen in the type II (ME49) strain. Rather than an effect on IFN-γ-dependent transcription, our data suggest that NTPase I was responsible for the strain-dependent difference in FLUC activity due to hydrolysis of ATP. We further show that NTPases I and II were not essential for tachyzoite growth in vitro or virulence in mice. Our study reveals that although T. gondii NTPases are not essential for immune evasion, they can affect ATP-dependent reporters. Importantly, this limitation was overcome using an ATP-independent Gaussia luciferase, which provides a more appropriate reporter for use with T. gondii infection studies., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Microneme secretion is essential for motility, invasion, and egress in apicomplexan parasites. Although previous studies indicate that Ca(2+) and cGMP control microneme secretion, little is known about how these pathways are naturally activated. Here we have developed genetically encoded indicators for Ca(2+) and microneme secretion to better define the signaling pathways that regulate these processes in Toxoplasma gondii We found that microneme secretion was triggered in vitro by exposure to a single host protein, serum albumin. The natural agonist serum albumin induced microneme secretion in a protein kinase G-dependent manner that correlated with increased cGMP levels. Surprisingly, serum albumin acted independently of elevated Ca(2+) and yet it was augmented by artificial agonists that raise Ca(2+), such as ethanol. Furthermore, although ethanol elevated intracellular Ca(2+), it alone was unable to trigger secretion without the presence of serum or serum albumin. This dichotomy was recapitulated by zaprinast, a phosphodiesterase inhibitor that elevated cGMP and separately increased Ca(2+) in a protein kinase G-independent manner leading to microneme secretion. Taken together, these findings reveal that microneme secretion is centrally controlled by protein kinase G and that this pathway is further augmented by elevation of intracellular Ca(2.), (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Calcium-dependent protein kinases (CDPKs) are expanded in apicomplexan parasites, especially in Toxoplasma gondii where 14 separate genes encoding these enzymes are found. Although previous studies have shown that several CDPKs play a role in controlling invasion, egress, and cell division in T. gondii, the roles of most of these genes are unexplored. Here we developed a more efficient method for gene disruption using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) that was modified to completely delete large, multiexonic genes from the genome and to allow serial replacement by recycling of the selectable marker using Cre-loxP. Using this system, we generated a total of 24 mutants in type 1 and 2 genetic backgrounds to ascertain the functions of noncanonical CDPKs. Remarkably, although we were able to confirm the essentiality of CDPK1 and CDPK7, the majority of CDPKs had no discernible phenotype for growth in vitro or infection in the mouse model. The exception to this was CDPK6, loss of which leads to reduced plaquing, fitness defect in a competition assay, and reduced tissue cyst formation in chronically infected mice. Our findings highlight the utility of CRISPR/Cas9 for rapid serial gene deletion and also suggest that additional models are needed to reveal the functions of many genes in T. gondii., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The protozoan parasite Toxoplasma gondii secretes a family of serine-threonine protein kinases into its host cell in order to disrupt signaling and alter immune responses. One prominent secretory effector is the rhoptry protein 18 (ROP18), a serine-threonine kinase that phosphorylates immunity related GTPases (IRGs) and hence blocks interferon gamma-mediated responses in rodent cells. Previous genetic studies show that ROP18 is a major virulence component of T. gondii strains from North and South America. Here, we implemented a high throughput screen to identify small molecule inhibitors of ROP18 in vitro and subsequently validated their specificity within infected cells. Although ROP18 was not susceptible to many kinase-directed inhibitors that affect mammalian kinases, the screen identified several sub micromolar inhibitors that belong to three chemical scaffolds: oxindoles, 6-azaquinazolines, and pyrazolopyridines. Treatment of interferon gamma-activated cells with one of these inhibitors enhanced immunity related GTPase recruitment to wild type parasites, recapitulating the defect of Δ rop18 mutant parasites, consistent with targeting ROP18 within infected cells. These compounds provide useful starting points for chemical biology experiments or as leads for therapeutic interventions designed to reduce parasite virulence.

Toxoplasma gondii is among the most prevalent parasites worldwide, infecting many wild and domestic animals and causing zoonotic infections in humans. T. gondii differs substantially in its broad distribution from closely related parasites that typically have narrow, specialized host ranges. To elucidate the genetic basis for these differences, we compared the genomes of 62 globally distributed T. gondii isolates to several closely related coccidian parasites. Our findings reveal that tandem amplification and diversification of secretory pathogenesis determinants is the primary feature that distinguishes the closely related genomes of these biologically diverse parasites. We further show that the unusual population structure of T. gondii is characterized by clade-specific inheritance of large conserved haploblocks that are significantly enriched in tandemly clustered secretory pathogenesis determinants. The shared inheritance of these conserved haploblocks, which show a different ancestry than the genome as a whole, may thus influence transmission, host range and pathogenicity.

Cryptosporidiosis is a serious diarrheal disease in immunocompromised patients and malnourished children, and treatment is complicated by a lack of adequate drugs. Recent studies suggest that the natural occurrence of a small gatekeeper residue in serine threonine calcium-dependent protein kinase 1 (CDPK1) of Cryptosporidium parvum might be exploited to target this enzyme and block parasite growth. Here were explored the potency with which a series of pyrazolopyrimidine analogs, which are selective for small gatekeeper kinases, inhibit C. parvum CDPK1 and block C. parvum growth in tissue culture in vitro. Although these compounds potently inhibited kinase activity in vitro, most had no effect on parasite growth. Moreover, among those that were active against parasite growth, there was a very poor correlation with their 50% inhibitory concentrations against the enzyme. Active compounds also had no effect on cell invasion, unlike the situation in Toxoplasma gondii, where these compounds block CDPK1, prevent microneme secretion, and disrupt cell invasion. These findings suggest that CPDK1 is not essential for C. parvum host cell invasion or growth and therefore that it is not the optimal target for therapeutic intervention. Nonetheless, several inhibitors with low micromolar 50% effective concentrations were identified, and these may affect other essential targets in C. parvum that are worthy of further exploration., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: A core set of autophagy proteins is required for gamma interferon (IFN-γ)-mediated clearance of Toxoplasma gondii in the mouse because of their control of several downstream effectors, including immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs). However, these effectors are absent (i.e., IRGs) from or nonessential (i.e., GBPs) in IFN-γ-activated human cells, raising the question of how these cells control parasite replication. Here, we define a novel role for ubiquitination and recruitment of autophagy adaptors in the strain-specific control of T. gondii replication in IFN-γ-activated human cells. Vacuoles containing susceptible strains of T. gondii became ubiquitinated, recruited the adaptors p62 and NDP52, and were decorated with LC3. Parasites within LC3-positive vacuoles became enclosed in multiple layers of host membranes, resulting in stunting of parasite replication. However, LC3-positive T. gondii-containing vacuoles did not fuse with endosomes and lysosomes, indicating that this process is fundamentally different from xenophagy, a form of autophagy involved in the control of intracellular bacterial pathogens. Genetic knockout of ATG16L or ATG7 reverted the membrane encapsulation and restored parasite replication, indicating that core autophagy proteins involved in LC3 conjugation are important in the control of parasite growth. Despite a role for the core autophagy machinery in this process, upstream activation through Beclin 1 was not sufficient to enhance the ubiquitination of T. gondii-containing vacuoles, suggesting a lack of reliance on canonical autophagy. These findings demonstrate a new mechanism for IFN-γ-dependent control of T. gondii in human cells that depends on ubiquitination and core autophagy proteins that mediate membrane engulfment and restricted growth., Importance: Autophagy is a process of cellular remodeling that allows the cell to recycle senescent organelles and recapture nutrients. During innate immune responses in the mouse, autophagy is recruited to help target intracellular pathogens and thus eliminate them. However, the antimicrobial mediators that depend on autophagy in the mouse are not conserved in humans, raising the issue of how human cells control intracellular pathogens. Our study defines a new pathway for the control of the ubiquitous intracellular parasite T. gondii in human cells activated by IFN-γ. Recruitment of autophagy adaptors resulted in engulfment of the parasite in multiple membranes and growth impairment. Although susceptible type 2 and 3 stains of T. gondii were captured by this autophagy-dependent pathway, type 1 strains were able to avoid entrapment., (Copyright © 2015 Selleck et al.)

Toxoplasma gondii has evolved a number of strategies to evade immune responses in its many hosts. Previous genetic mapping of crosses between clonal type 1, 2, and 3 strains of T. gondii, which are prevalent in Europe and North America, identified two rhoptry proteins, ROP5 and ROP18, that function together to block innate immune mechanisms activated by interferon gamma (IFNg) in murine hosts. However, the contribution of these and other virulence factors in more genetically divergent South American strains is unknown. Here we utilized a cross between the intermediately virulent North American type 2 ME49 strain and the highly virulent South American type 10 VAND strain to map the genetic basis for differences in virulence in the mouse. Quantitative trait locus (QTL) analysis of this new cross identified one peak that spanned the ROP5 locus on chromosome XII. CRISPR-Cas9 mediated deletion of all copies of ROP5 in the VAND strain rendered it avirulent and complementation confirmed that ROP5 is the major virulence factor accounting for differences between type 2 and type 10 strains. To extend these observations to other virulent South American strains representing distinct genetic populations, we knocked out ROP5 in type 8 TgCtBr5 and type 4 TgCtBr18 strains, resulting in complete loss of virulence in both backgrounds. Consistent with this, polymorphisms that show strong signatures of positive selection in ROP5 were shown to correspond to regions known to interface with host immunity factors. Because ROP5 and ROP18 function together to resist innate immune mechanisms, and a significant interaction between them was identified in a two-locus scan, we also assessed the role of ROP18 in the virulence of South American strains. Deletion of ROP18 in South American type 4, 8, and 10 strains resulted in complete attenuation in contrast to a partial loss of virulence seen for ROP18 knockouts in previously described type 1 parasites. These data show that ROP5 and ROP18 are conserved virulence factors in genetically diverse strains from North and South America, suggesting they evolved to resist innate immune defenses in ancestral T. gondii strains, and they have subsequently diversified under positive selection.

Apicomplexan parasites cause some of the most severe human diseases, including malaria (caused by Plasmodium), toxoplasmosis, and cryptosporidiosis. Treatments are limited by the lack of effective drugs and development of resistance to available agents. By exploiting novel features of protein kinases in these parasites, it may be possible to develop new treatments. We summarize here recent advances in identifying small molecule inhibitors against a novel family of plant-like, calcium-dependent kinases that are uniquely expanded in apicomplexan parasites. Analysis of the 3D structure, activation mechanism, and sensitivity to small molecules had identified several attractive chemical scaffolds that are potent and selective inhibitors of these parasite kinases. Further optimization of these leads may yield promising new drugs for treatment of these parasitic infections., (Copyright © 2015 Elsevier Ltd. All rights reserved.)