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The population genetic structure of Toxoplasma gondii was determined by multilocus restriction fragment length polymorphism analysis at 6 loci in 106 independent isolates from humans and animals. Phylogenetic and statistical analyses indicated a highly unusual population structure consisting of 3 widespread clonal lineages. Extensively mixed genotypes were only apparent in 4 strains, which indicated that, while not separate species, sexual recombination between the 3 lineages is exceedingly rare in natural populations. T. gondii is a major cause of subclinical human infection and an important opportunistic pathogen that causes severe disease in immunocompromised patients. While strains from all 3 lineages were isolated from humans, the majority of human toxoplasmosis cases were associated with strains of a type II genotype. The correlation of specific clonal lineages with human toxoplasmosis has important implications for development of vaccines, drug treatments, and diagnostic protocols. [ABSTRACT FROM AUTHOR]

In both mice and humans, Type II interferon gamma (IFNγ) is crucial for the regulation of Toxoplasma gondii ( T. gondii ) infection, during acute or chronic phases. To thwart this defense, T. gondii secretes protein effectors hindering the host's immune response. For example, T. gondii relies on the MYR translocon complex to deploy soluble dense granule effectors (GRAs) into the host cell cytosol or nucleus. Recent genome-wide loss-of-function screens in IFNγ-primed primary human fibroblasts identified MYR translocon components as crucial for parasite resistance against IFNγ-driven vacuole clearance. However, these screens did not pinpoint specific MYR-dependent GRA proteins responsible for IFNγ signaling blockade, suggesting potential functional redundancy. Our study reveals that T. gondii depends on the MYR translocon complex to prevent parasite premature egress and host cell death in human cells stimulated with IFNγ post-infection, a unique phenotype observed in various human cell lines but not in murine cells. Intriguingly, inhibiting parasite egress did not prevent host cell death, indicating this mechanism is distinct from those described previously. Genome-wide loss-of-function screens uncovered TgIST, GRA16, GRA24, and GRA28 as effectors necessary for a complete block of IFNγ response. GRA24 and GRA28 directly influenced IFNγ-driven transcription, GRA24's action depended on its interaction with p38 MAPK, while GRA28 disrupted histone acetyltransferase activity of CBP/p300. Given the intricate nature of the immune response to T. gondii , it appears that the parasite has evolved equally elaborate mechanisms to subvert IFNγ signaling, extending beyond direct interference with the JAK/STAT1 pathway, to encompass other signaling pathways as well.IMPORTANCE Toxoplasma gondii , an intracellular parasite, affects nearly one-third of the global human population, posing significant risks for immunocompromised patients and infants infected in utero . In murine models, the core mechanisms of IFNγ-mediated immunity against T. gondii are consistently preserved, showcasing a remarkable conservation of immune defense mechanisms. In humans, the recognized restriction mechanisms vary among cell types, lacking a universally applicable mechanism. This difference underscores a significant variation in the genes employed by T. gondii to shield itself against the IFNγ response in human vs murine cells. Here, we identified a specific combination of four parasite-secreted effectors deployed into the host cell nucleus, disrupting IFNγ signaling. This disruption is crucial in preventing premature egress of the parasite and host cell death. Notably, this phenotype is exclusive to human cells, highlighting the intricate and unique mechanisms T. gondii employs to modulate host responses in the human cellular environment., Competing Interests: The authors declare no conflict of interest.

Biological studies of the determinants of Cryptosporidium infectivity are lacking despite the fact that cryptosporidiosis is a major public health problem. Recently, the 60-kDa glycoprotein (GP60) has received attention because of its high sequence polymorphism and association with host infectivity of isolates and protection against reinfection. However, studies of GP60 function have been hampered by its heavy O-linked glycosylation. Here, we used advanced genetic tools to investigate the processing, fate, and function of GP60. Endogenous gene tagging showed that the GP60 cleavage products, GP40 and GP15, are both highly expressed on the surface of sporozoites, merozoites and male gametes. During invasion, GP40 translocates to the apical end of the zoites and remains detectable at the parasite-host interface. Deletion of the signal peptide, GPI anchor, and GP15 sequences affects the membrane localization of GP40. Deletion of the GP60 gene significantly reduces parasite growth and severity of infection, and replacement of the GP60 gene with sequence from an avirulent isolate reduces the pathogenicity of a highly infective isolate. These results have revealed dynamic changes in GP60 expression during parasite development. They further suggest that GP60 is a key protein mediating host infectivity and pathogenicity., (© 2024. The Author(s).)

Toxoplasma gondii bradyzoites play a critical role in pathology due to their long-term persistence in intermediate hosts and their potential to reactivate, resulting in severe diseases in immunocompromised individuals. Currently, there is no effective treatment for eliminating bradyzoites. Hence, better in vitro models of T. gondii bradyzoite development would facilitate identification of therapeutic targets for bradyzoites. Herein, we characterized a natural isolate of T. gondii , called Tg68, which showed slower in vitro replication of tachyzoites, and permissive bradyzoite development under stress conditions in vitro . Transcriptional analysis revealed constitutive expression in Tg68 tachyzoites of the key regulators of bradyzoite development including BFD1 , BFD2 , and several AP2 factors. Consistent with this finding, Tg68 tachyzoites expressed high levels of bradyzoite-specific genes including BAG1 , ENO1 , and LDH2 . Moreover, after stress-induced differentiation, Tg68 bradyzoites exhibited gene expression profiles of mature bradyzoites, even at early time points. These data suggest that Tg68 tachyzoites exist in a pre-bradyzoite stage primed to readily develop into mature bradyzoites under stress conditions in vitro . Tg68 presents a novel model for differentiation in vitro that will serve as a useful tool for the investigation of bradyzoite biology and the development of therapeutics., Importance: Toxoplasma gondii is a widespread protozoan that chronically infects ~30% of the world's population. T. gondii can differentiate between the fast-growing life stage that causes acute infection and the slow-growing stage that persists in the host for extended periods of time. The slow-growing stage cannot be eliminated by the host immune response or currently known antiparasitic drugs. Studies on the slow-growing stage have been limited due to the limitations of in vivo experiments and the challenges of in vitro manipulation. Here, we characterize a natural isolate of T. gondii , which constitutively expresses factors that drive development and that is permissive to convert to the slow-growing stage under stress conditions in vitro . The strain presents a novel in vitro model for studying the chronic phase of toxoplasmosis and identifying new therapeutic treatments for chronic infections., Competing Interests: The authors declare no conflict of interest.

Toxoplasma gondii causes widespread chronic infections that are not cured by current treatments due to inability to affect semi-dormant bradyzoite stages within tissue cysts. To identify compounds to eliminate chronic infection, we developed a HTS using a recently characterized strain of T. gondii that undergoes efficient conversion to bradyzoites in intro. Stage-specific expression of luciferase was used to selectively monitor growth inhibition of bradyzoites by the Library of Pharmacological Active Compounds, consisting of 1,280 drug-like compounds. We identified 44 compounds with >50% inhibitory effects against bradyzoites, including new highly potent compounds, several of which have precedent for antimicrobial activity. Subsequent characterization of the compound Sanguinarine sulfate revealed potent and rapid killing against in vitro produced bradyzoites and bradyzoites harvested from chronically infected mice. These findings provide a platform for expanded screening and identify promising compounds for further preclinical development against T. gondii bradyzoites responsible for chronic infection.

Very little is known about the process of meiosis in the apicomplexan parasite Cryptosporidium despite the essentiality of sex in its life cycle. Most cell lines only support asexual growth of Cryptosporidium parvum (C. parvum), but stem cell derived intestinal epithelial cells grown under air-liquid interface (ALI) conditions support the sexual cycle. To examine chromosomal dynamics during meiosis in C. parvum, we generated two transgenic lines of parasites that were fluorescently tagged with mCherry or GFP on chromosomes 1 or 5, respectively. Infection of ALI cultures or Ifngr1-/- mice with mCherry and GFP parasites resulted in cross-fertilization and the formation of "yellow" oocysts, which contain 4 haploid sporozoites that are the product of meiosis. Recombinant oocysts from the F1 generation were purified and used to infect HCT-8 cultures, and phenotypes of the progeny were observed by microscopy. All possible phenotypes predicted by independent segregation were represented equally (~25%) in the population, indicating that C. parvum chromosomes exhibit a Mendelian inheritance pattern. The most common pattern observed from the outgrowth of single oocysts included all possible parental and recombinant phenotypes derived from a single meiotic event, suggesting a high rate of crossover. To estimate the frequency of crossover, additional loci on chromosomes 1 and 5 were tagged and used to monitor intrachromosomal crosses in Ifngr1-/- mice. Both chromosomes showed a high frequency of crossover compared to other apicomplexans with map distances (i.e., 1% recombination) of 3-12 kb. Overall, a high recombination rate may explain many unique characteristics observed in Cryptosporidium spp. such as high rates of speciation, wide variation in host range, and rapid evolution of host-specific virulence factors., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kimball et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Previous studies have shown that bicyclic azetidines are potent and selective inhibitors of apicomplexan phenylalanine tRNA synthetase (PheRS), leading to parasite growth inhibition in vitro and in vivo, including in models of Toxoplasma infection. Despite these useful properties, additional optimization is required for the development of efficacious treatments of toxoplasmosis from this inhibitor series, in particular, to achieve optimal exposure in the brain. Here, we describe a series of PheRS inhibitors built on a new bicyclic pyrrolidine core scaffold designed to retain the exit-vector geometry of the isomeric bicyclic azetidine core scaffold while offering avenues to sample diverse chemical space. Relative to the parent series, bicyclic pyrrolidines retain reasonable potency and target selectivity for parasite PheRS vs host. Further structure-activity relationship studies revealed that the introduction of aliphatic groups improved potency and ADME and PK properties, including brain exposure. The identification of this new scaffold provides potential opportunities to extend the analogue series to further improve selectivity and potency and ultimately deliver a novel, efficacious treatment of toxoplasmosis.

Importance: Autophagy is a process used by cells to recycle organelles and macromolecules and to eliminate intracellular pathogens. Previous studies have shown that some stains of Toxoplasma gondii are resistant to autophagy-dependent growth restriction, while others are highly susceptible. Although it is known that autophagy-mediated control requires activation by interferon gamma, the basis for why parasite strains differ in their susceptibility is unknown. Our findings indicate that susceptibility involves at least five unlinked parasite genes on different chromosomes, including several secretory proteins targeted to the parasite-containing vacuole and exposed to the host cell cytosol. Our findings reveal that susceptibility to autophagy-mediated growth restriction relies on differential recognition of parasite proteins exposed at the host-pathogen interface, thus identifying a new mechanism for cell-autonomous control of intracellular pathogens., Competing Interests: The authors declare no conflict of interest.

Cryptosporidium parvum is an obligate intracellular parasite with a highly reduced mitochondrion that lacks the tricarboxylic acid cycle and the ability to generate ATP, making the parasite reliant on glycolysis. Genetic ablation experiments demonstrated that neither of the two putative glucose transporters CpGT1 and CpGT2 were essential for growth. Surprisingly, hexokinase was also dispensable for parasite growth while the downstream enzyme aldolase was required, suggesting the parasite has an alternative way of obtaining phosphorylated hexose. Complementation studies in E. coli support a role for direct transport of glucose-6-phosphate from the host cell by the parasite transporters CpGT1 and CpGT2, thus bypassing a requirement for hexokinase. Additionally, the parasite obtains phosphorylated glucose from amylopectin stores that are released by the action of the essential enzyme glycogen phosphorylase. Collectively, these findings reveal that C. parvum relies on multiple pathways to obtain phosphorylated glucose both for glycolysis and to restore carbohydrate reserves., (© 2024. The Author(s).)

To define cellular immunity to the intracellular pathogen Toxoplasma gondii , we performed a genome-wide CRISPR loss-of-function screen to identify genes important for (interferon gamma) IFN-γ-dependent growth restriction. We revealed a role for the tumor suppressor NF2/Merlin for maximum induction of Interferon Stimulated Genes (ISG), which are positively regulated by the transcription factor IRF-1. We then performed an ISG-targeted CRISPR screen that identified the host E3 ubiquitin ligase RNF213 as necessary for IFN-γ-mediated control of T. gondii in multiple human cell types. RNF213 was also important for control of bacterial ( Mycobacterium tuberculosis ) and viral (Vesicular Stomatitis Virus) pathogens in human cells. RNF213-mediated ubiquitination of the parasitophorous vacuole membrane (PVM) led to growth restriction of T. gondii in response to IFN-γ. Moreover, overexpression of RNF213 in naive cells also impaired growth of T. gondii . Surprisingly, growth inhibition did not require the autophagy protein ATG5, indicating that RNF213 initiates restriction independent of a previously described noncanonical autophagy pathway. Mutational analysis revealed that the ATPase domain of RNF213 was required for its recruitment to the PVM, while loss of a critical histidine in the RZ finger domain resulted in partial reduction of recruitment to the PVM and complete loss of ubiquitination. Both RNF213 mutants lost the ability to restrict growth of T. gondii , indicating that both recruitment and ubiquitination are required. Collectively, our findings establish RNF213 as a critical component of cell-autonomous immunity that is both necessary and sufficient for control of intracellular pathogens in human cells., Competing Interests: Competing interests statement:The authors declare no competing interest.

Cryptosporidiosis is a major cause of severe diarrheal disease in infants from resource poor settings. The majority of infections are caused by the human-specific pathogen C. hominis and absence of in vitro growth platforms has limited our understanding of host-pathogen interactions and development of effective treatments. To address this problem, we developed a stem cell-derived culture system for C. hominis using human enterocytes differentiated under air-liquid interface (ALI) conditions. Human ALI cultures supported robust growth and complete development of C. hominis in vitro including all life cycle stages. Cryptosporidium infection induced a strong interferon response from enterocytes, possibly driven, in part, by an endogenous dsRNA virus in the parasite. Prior infection with Cryptosporidium induced type III IFN secretion and consequently blunted infection with Rotavirus, including live attenuated vaccine strains. The development of hALI provides a platform for further studies on human-specific pathogens, including clinically important coinfections that may alter vaccine efficacy.

Importance: Interferon-γ (IFNγ) is a critical mediator of cell-intrinsic immunity to intracellular pathogens. Understanding the complex cellular mechanisms supporting robust interferon-γ-induced host defenses could aid in developing new therapeutics to treat infections. Here, we examined the impact of autophagy genes in the interferon-γ-induced host response. We demonstrate that genes within the autophagy pathway including Wipi2 , Atg9 , and Gate-16 , as well as ubiquitin ligase complex genes Cul3 and Klhl9 are required for IFNγ-induced inhibition of murine norovirus (norovirus hereinafter) replication in mouse cells. WIPI2 and GATE-16 were also required for IFNγ-mediated restriction of parasite growth within the Toxoplasma gondii parasitophorous vacuole in human cells. Furthermore, we found that perturbation of UFMylation pathway components led to more robust IFNγ-induced inhibition of norovirus via regulation of endoplasmic reticulum (ER) stress. Enhancing or inhibiting these dynamic cellular components could serve as a strategy to control intracellular pathogens and maintain an effective immune response., Competing Interests: D.R.B., M.R.M., A.P., S.H., and H.W.V. are employees of and hold stock in Vir Biotechnology, where some of the work was performed. D.R.B., M.R.M., A.P., and H.W.V. made the initial observations reported here while at Washington University School of Medicine in St. Louis without funding from Vir Biotechnology. H.W.V. is a founder of Casma Therapeutics and PiernianDx, neither of which funded the research reported herein. L.D.S. is a consultant for Kainomyx, which had no role in the study.

Cryptosporidiosis is a leading cause of life-threatening diarrhea in young children in resource-poor settings. To explore microbial influences on susceptibility, we screened 85 microbiota-associated metabolites for their effects on Cryptosporidium parvum growth in vitro. We identify eight inhibitory metabolites in three main classes: secondary bile salts/acids, a vitamin B 6 precursor, and indoles. Growth restriction of C. parvum by indoles does not depend on the host aryl hydrocarbon receptor (AhR) pathway. Instead, treatment impairs host mitochondrial function and reduces total cellular ATP, as well as directly reducing the membrane potential in the parasite mitosome, a degenerate mitochondria. Oral administration of indoles, or reconstitution of the gut microbiota with indole-producing bacteria, delays life cycle progression of the parasite in vitro and reduces the severity of C. parvum infection in mice. Collectively, these findings indicate that microbiota metabolites impair mitochondrial function and contribute to colonization resistance to Cryptosporidium infection., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Host immune responses are required for the efficient control of cryptosporidiosis. Immunity against Cryptosporidium infection has been best studied in mice, where it is mediated by both innate and adaptive immune responses. Dendritic cells are the key link between innate and adaptive immunity and participate in the defense against Cryptosporidium infection. While the effector mechanism varies, both humans and mice rely on dendritic cells for sensing parasites and restricting infection. Recently, the use of mouse-adapted strains C. parvum and mouse-specific strain C. tyzzeri have provided tractable systems to study the role of dendritic cells in mice against this parasite. In this review, we provide an overview of recent advances in innate immunity acting during infection with Cryptosporidium with a major focus on the role of dendritic cells in the intestinal mucosa. Further work is required to understand the role of dendritic cells in the activation of T cells and to explore associated molecular mechanisms. The identification of Cryptosporidium antigen involved in the activation of Toll-like receptor signaling in dendritic cells during infection is also a matter of future study. The in-depth knowledge of immune responses in cryptosporidiosis will help develop targeted prophylactic and therapeutic interventions.

Cryptosporidium parvum is an enteric pathogen that invades epithelial cells in the intestine, where it resides at the apical surface in a unique epicellular location. Compared with those of related apicomplexan parasites, the processes of host cell attachment and invasion by C. parvum are poorly understood. The streamlined C. parvum genome contains numerous mucin-like glycoproteins, several of which have previously been shown to mediate cell attachment, although the majority are unstudied. Here, we identified the antigens recognized by monoclonal antibody (MAb) 1A5, which stains the apical end of sporozoites and mature merozoites. Immunoprecipitation with MAb 1A5 followed by mass spectrometry identified a heterodimer comprised of paralogous proteins which are related to additional orthologs in the genome of C. parvum and related species. Paralogous glycoproteins recognized by MAb 1A5 heterodimerize as a complex displayed on the parasite surface, and they also interact with lectins that suggest that they contain mucin-like, O-linked oligosaccharides. Although the gene encoding one of the paralogs was readily disrupted by CRISPR/Cas9 gene editing, its partner, which contains a mucin-like domain related to GP900, was refractory to deletion. Combined with the ability of MAb 1A5 to partially neutralize host cell attachment by sporozoites, these findings define a new family of secretory glycoproteins that participate in cell invasion by Cryptosporidium spp. IMPORTANCE Although Cryptosporidium is extremely efficient at penetrating mucus and invading epithelial cells in the intestine, the mechanism of cell attachment is poorly understood. To expand our understanding of this process, we characterized the antigens recognized by a monoclonal antibody that stains the apical end of invasive stages called sporozoites and merozoites. Our studies identify a family of glycoproteins that form heterodimers on the parasite cell surface to facilitate host cell attachment and entry. By further defining the role of mucin-like glycoproteins in host cell attachment, our studies may lead to strategies to disrupt cell adhesion and thereby decrease infection.

Apicomplexan parasite growth and replication relies on nutrient acquisition from host cells, in which intracellular multiplication occurs, yet the mechanisms that underlie the nutrient salvage remain elusive. Numerous ultrastructural studies have documented a plasma membrane invagination with a dense neck, termed the micropore, on the surface of intracellular parasites. However, the function of this structure remains unknown. Here we validate the micropore as an essential organelle for endocytosis of nutrients from the host cell cytosol and Golgi in the model apicomplexan Toxoplasma gondii. Detailed analyses demonstrated that Kelch13 is localized at the dense neck of the organelle and functions as a protein hub at the micropore for endocytic uptake. Intriguingly, maximal activity of the micropore requires the ceramide de novo synthesis pathway in the parasite. Thus, this study provides insights into the machinery underlying acquisition of host cell-derived nutrients by apicomplexan parasites that are otherwise sequestered from host cell compartments., (© 2023. The Author(s).)

Phenotypic switching between tachyzoite and bradyzoite is the fundamental mechanism underpinning the pathogenicity and adaptability of the protozoan parasite Toxoplasma gondii. Although accumulation of cytoplasmic starch granules is a hallmark of the quiescent bradyzoite stage, the regulatory factors and mechanisms contributing to amylopectin storage in bradyzoites are incompletely known. Here, we show that T. gondii protein phosphatase 2A (PP2A) holoenzyme is composed of a catalytic subunit PP2A-C, a scaffold subunit PP2A-A and a regulatory subunit PP2A-B. Disruption of any of these subunits increased starch accumulation and blocked the tachyzoite-to-bradyzoite differentiation. PP2A contributes to the regulation of amylopectin metabolism via dephosphorylation of calcium-dependent protein kinase 2 at S679. Phosphoproteomics identified several putative PP2A holoenzyme substrates that are involved in bradyzoite differentiation. Our findings provide novel insight into the role of PP2A as a key regulator of starch metabolism and bradyzoite differentiation in T. gondii., (© 2022. The Author(s).)

Signal transducer and activator of transcription (STAT) proteins communicate from cell-surface receptors to drive transcription of immune response genes. The parasite Toxoplasma gondii blocks STAT1-mediated gene expression by secreting the intrinsically disordered protein TgIST that traffics to the host nucleus, binds phosphorylated STAT1 dimers, and occupies nascent transcription sites that unexpectedly remain silenced. Here we define a core region within internal repeats of TgIST that is necessary and sufficient to block STAT1-mediated gene expression. Cellular, biochemical, mutational, and structural data demonstrate that the repeat region of TgIST adopts a helical conformation upon binding to STAT1 dimers. The binding interface is defined by a groove formed from two loops in the STAT1 SH2 domains that reorient during dimerization. TgIST binding to this newly exposed site at the STAT1 dimer interface alters its conformation and prevents the recruitment of co-transcriptional activators, thus defining the mechanism of blocked transcription., (© 2022. The Author(s).)

Given the importance of epigenetic modification, pathogens have found a variety of ways to alter chromatin and affect host gene expression. The apicomplexan parasite Toxoplasma gondii expresses two nuclear targeted secreted effectors TgIST and TgNSM that target the activity of host histone deacetylase regulating corepressor complexes NuRD and NCoR/SMRT, respectively. TgIST and TgNSM are crucial for blocking the host interferon response protecting both the acute and latent stages of the infection. T. gondii represents a unique model organism to study the significance of epigenetic modifications in the regulation of interferon responses and other transcriptional responses at the interface of host-pathogen interaction.

Toxoplasma gondii commonly infects humans and while most infections are controlled by the immune response, currently approved drugs are not capable of clearing chronic infection in humans. Hence, approximately one third of the world's human population is at risk of reactivation, potentially leading to severe sequelae. To identify new candidates for treating chronic infection, we investigated a series of compounds derived from diversity-oriented synthesis. Bicyclic azetidines are potent low nanomolar inhibitors of phenylalanine tRNA synthetase (PheRS) in T. gondii, with excellent selectivity. Biochemical and genetic studies validate PheRS as the primary target of bicyclic azetidines in T. gondii, providing a structural basis for rational design of improved analogs. Favorable pharmacokinetic properties of a lead compound provide excellent protection from acute infection and partial protection from chronic infection in an immunocompromised mouse model of toxoplasmosis. Collectively, PheRS inhibitors of the bicyclic azetidine series offer promise for treatment of chronic toxoplasmosis., (© 2022. The Author(s).)

Autophagy is a highly conserved process that utilizes lysosomes to selectively degrade a variety of intracellular cargo, thus providing quality control over cellular components and maintaining cellular regulatory functions. Autophagy is triggered by multiple stimuli ranging from nutrient starvation to microbial infection. Autophagy extensively shapes and modulates the inflammatory response, the concerted action of immune cells, and secreted mediators aimed to eradicate a microbial infection or to heal sterile tissue damage. Here, we first review how autophagy affects innate immune signaling, cell-autonomous immune defense, and adaptive immunity. Then, we discuss the role of non-canonical autophagy in microbial infections and inflammation. Finally, we review how crosstalk between autophagy and inflammation influences infectious, metabolic, and autoimmune disorders.

Toxoplasma gondii is an important human pathogen infecting an estimated one in three people worldwide. The cytokine interferon gamma (IFNγ) is induced during infection and is critical for restricting T. gondii growth in human cells. Growth restriction is presumed to be due to the induction of interferon-stimulated genes (ISGs) that are upregulated to protect the host from infection. Although there are hundreds of ISGs induced by IFNγ, their individual roles in restricting parasite growth in human cells remain somewhat elusive. To address this deficiency, we screened a library of 414 IFNγ induced ISGs to identify factors that impact T. gondii infection in human cells. In addition to IRF1, which likely acts through the induction of numerous downstream genes, we identified RARRES3 as a single factor that restricts T. gondii infection by inducing premature egress of the parasite in multiple human cell lines. Overall, while we successfully identified a novel IFNγ induced factor restricting T. gondii infection, the limited number of ISGs capable of restricting T. gondii infection when individually expressed suggests that IFNγ-mediated immunity to T. gondii infection is a complex, multifactorial process., Competing Interests: NR, MA, SM, NA, LS No competing interests declared, JS Reviewing editor, eLife, (© 2021, Rinkenberger et al.)

Toxoplasma gondii has evolved different developmental stages for disseminating during acute infection (i.e., tachyzoites) and establishing chronic infection (i.e., bradyzoites). Calcium ion (Ca 2+ ) signaling tightly regulates the lytic cycle of tachyzoites by controlling microneme secretion and motility to drive egress and cell invasion. However, the roles of Ca 2+ signaling pathways in bradyzoites remain largely unexplored. Here, we show that Ca 2+ responses are highly restricted in bradyzoites and that they fail to egress in response to agonists. Development of dual-reporter parasites revealed dampened Ca 2+ responses and minimal microneme secretion by bradyzoites induced in vitro or harvested from infected mice and tested ex vivo. Ratiometric Ca 2+ imaging demonstrated lower Ca 2+ basal levels, reduced magnitude, and slower Ca 2+ kinetics in bradyzoites compared with tachyzoites stimulated with agonists. Diminished responses in bradyzoites were associated with downregulation of Ca 2+ -ATPases involved in intracellular Ca 2+ storage in the endoplasmic reticulum (ER) and acidocalcisomes. Once liberated from cysts by trypsin digestion, bradyzoites incubated in glucose plus Ca 2+ rapidly restored their intracellular Ca 2+ and ATP stores, leading to enhanced gliding. Collectively, our findings indicate that intracellular bradyzoites exhibit dampened Ca 2+ signaling and lower energy levels that restrict egress, and yet upon release they rapidly respond to changes in the environment to regain motility., Competing Interests: YF, KB, NJ, SM, LS No competing interests declared, (© 2021, Fu et al.)

Cryptosporidium can cause severe diarrhea and morbidity, but many infections are asymptomatic. Here, we studied the immune response to a commensal strain of Cryptosporidium tyzzeri (Ct-STL) serendipitously discovered when conventional type 1 dendritic cell (cDC1)-deficient mice developed cryptosporidiosis. Ct-STL was vertically transmitted without negative health effects in wild-type mice. Yet, Ct-STL provoked profound changes in the intestinal immune system, including induction of an IFN-γ-producing Th1 response. TCR sequencing coupled with in vitro and in vivo analysis of common Th1 TCRs revealed that Ct-STL elicited a dominant antigen-specific Th1 response. In contrast, deficiency in cDC1s skewed the Ct-STL CD4 T cell response toward Th17 and regulatory T cells. Although Ct-STL predominantly colonized the small intestine, colon Th1 responses were enhanced and associated with protection against Citrobacter rodentium infection and exacerbation of dextran sodium sulfate and anti-IL10R-triggered colitis. Thus, Ct-STL represents a commensal pathobiont that elicits Th1-mediated intestinal homeostasis that may reflect asymptomatic human Cryptosporidium infection., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Oral infection with Toxoplasma gondii results in dysbiosis and enteritis, both of which revert to normal during chronic infection. However, whether infection leaves a lasting impact on mucosal responses remains uncertain. Here we examined the effect of the chemical irritant dextran sodium sulfate (DSS) on intestinal damage and wound healing in chronically infected mice. Our findings indicate that prior infection with T. gondii exacerbates damage to the colon caused by DSS and impairs wound healing by suppressing stem cell regeneration of the epithelium. Enhanced tissue damage was attributable to inflammatory monocytes that emerge preactivated from bone marrow, migrate to the intestine, and release inflammatory mediators, including nitric oxide. Tissue damage was reversed by neutralization of inflammatory monocytes or nitric oxide, revealing a causal mechanism for tissue damage. Our findings suggest that chronic infection with T. gondii enhances monocyte activation to increase inflammation associated with a secondary environmental insult., Competing Interests: The authors declare no competing interest.

Toxoplasma gondii translocates effector proteins into its host cell to subvert various host pathways. T. gondii effector TgIST blocks the transcription of interferon-stimulated genes to reduce immune defense. Interferons upregulate numerous genes, including protein kinase R (PKR), which induce necrosome formation to activate mixed-lineage-kinase-domain-like (MLKL) pseudokinase and induce necroptosis. Whether these interferon functions are targeted by Toxoplasma is unknown. Here, we examine secreted effectors that localize to the host cell nucleus and find that the chronic bradyzoite stage secretes effector TgNSM that targets the NCoR/SMRT complex, a repressor for various transcription factors, to inhibit interferon-regulated genes involved in cell death. TgNSM acts with TgIST to block IFN-driven expression of PKR and MLKL, thus preventing host cell necroptotic death and protecting the parasite's intracellular niche. The mechanism of action of TgNSM uncovers a role of NCoR/SMRT in necroptosis, assuring survival of intracellular cysts and chronic infection., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Toxoplasma gondii is a parasite that infects a wide range of animals and causes zoonotic infections in humans. Although it normally only results in mild illness in healthy individuals, toxoplasmosis is a common opportunistic infection with high mortality in individuals who are immunocompromised, most commonly due to reactivation of infection in the central nervous system. In the acute phase of infection, interferon-dependent immune responses control rapid parasite expansion and mitigate acute disease symptoms. However, after dissemination the parasite differentiates into semi-dormant cysts that form within muscle cells and neurons, where they persist for life in the infected host. Control of infection in the central nervous system, a compartment of immune privilege, relies on modified immune responses that aim to balance infection control while limiting potential damage due to inflammation. In response to the activation of interferon-mediated pathways, the parasite deploys an array of effector proteins to escape immune clearance and ensure latent survival. Although these pathways are best studied in the laboratory mouse, emerging evidence points to unique mechanisms of control in human toxoplasmosis. In this Review, we explore some of these recent findings that extend our understanding for proliferation, establishment and control of toxoplasmosis in humans.

In living cells, microtubules (MTs) play pleiotropic roles, which require very different mechanical properties. Unlike the dynamic MTs found in the cytoplasm of metazoan cells, the specialized cortical MTs from Toxoplasma gondii, a prevalent human pathogen, are extraordinarily stable and resistant to detergent and cold treatments. Using single-particle cryo-EM, we determine their ex vivo structure and identify three proteins (TrxL1, TrxL2 and SPM1) as bona fide microtubule inner proteins (MIPs). These three MIPs form a mesh on the luminal surface and simultaneously stabilize the tubulin lattice in both longitudinal and lateral directions. Consistent with previous observations, deletion of the identified MIPs compromises MT stability and integrity under challenges by chemical treatments. We also visualize a small molecule like density at the Taxol-binding site of β-tubulin. Our results provide the structural basis to understand the stability of cortical MTs and suggest an evolutionarily conserved mechanism of MT stabilization from the inside.

The apicomplexan parasite Cryptosporidium parvum contains an expanded family of 22 insulinase-like proteases (INS), a feature that contrasts with their otherwise streamlined genome. Here, we examined the function of INS1, which is most similar to the human insulinase protease that cleaves a variety of small peptide substrates. INS1 is an M16A clan member and contains a signal peptide, an N-terminal domain with the HXXEH active site, followed by three inactive domains. Unlike previously studied C. parvum INS proteins that are expressed in sporozoites and during merogony, INS1 was expressed exclusively in macrogamonts, where it was localized in small cytoplasmic vesicles. Although INS1 did not colocalize with the oocyst wall protein recognized by the antibody OW50, immune-electron microscopy indicated that INS1 resides in small vesicles in the secretory system. Notably, these small INS1-positive vesicles were often in close proximity to large OW50-positive vacuoles resembling wall-forming bodies, which contain precursors for oocyst wall formation. Genetic deletion of INS1, or replacement with an active-site mutant, resulted in lower formation of macrogamonts in vitro and reduced oocyst shedding in vivo Our findings reveal that INS1 functions in the formation or maturation of macrogamonts and that its loss results in attenuated virulence in immunocompromised mice. IMPORTANCE Cryptosporidiosis is a debilitating diarrheal disease in young children in developing countries. The absence of effective treatments or vaccines makes this infection very difficult to manage in susceptible populations. Although the oral dose of oocysts needed to cause infection is low, infected individuals shed very high numbers of oocysts, readily contaminating the environment. Our studies demonstrate that the protease INS1 is important for formation of female sexual stages and that in its absence, parasites produce fewer oocysts and are attenuated in immunocompromised mice. These findings suggest that mutants lacking INS1, or related proteases, are useful for further characterizing the pathway that leads to macrogamont maturation and oocyst wall formation., (Copyright © 2021 Xu et al.)

The protozoan parasite Cryptosporidium sp. is a leading cause of diarrheal disease in those with compromised or underdeveloped immune systems, particularly infants and toddlers in resource-poor localities. As an enteric pathogen, Cryptosporidium sp. invades the apical surface of intestinal epithelial cells, where it resides in close proximity to metabolites in the intestinal lumen. However, the effect of gut metabolites on susceptibility to Cryptosporidium infection remains largely unstudied. Here, we first identified which gut metabolites are prevalent in neonatal mice when they are most susceptible to Cryptosporidium parvum infection and then tested the isolated effects of these metabolites on C. parvum invasion and growth in intestinal epithelial cells. Our findings demonstrate that medium or long-chain saturated fatty acids inhibit C. parvum growth, perhaps by negatively affecting the streamlined metabolism in C. parvum , which is unable to synthesize fatty acids. Conversely, long-chain unsaturated fatty acids enhanced C. parvum invasion, possibly by modulating membrane fluidity. Hence, gut metabolites, either from diet or produced by the microbiota, influence C. parvum growth in vitro and may also contribute to the early susceptibility to cryptosporidiosis seen in young animals. IMPORTANCE Cryptosporidium sp. occupies a unique intracellular niche that exposes the parasite to both host cell contents and the intestinal lumen, including metabolites from the diet and produced by the microbiota. Both dietary and microbial products change over the course of early development and could contribute to the changes seen in susceptibility to cryptosporidiosis in humans and mice. Consistent with this model, we show that the immature gut metabolome influenced the growth of Cryptosporidium parvum in vitro Interestingly, metabolites that significantly altered parasite growth were fatty acids, a class of molecules that Cryptosporidium sp. is unable to synthesize de novo The enhancing effects of polyunsaturated fatty acids and the inhibitory effects of saturated fatty acids presented in this study may provide a framework for future studies into this enteric parasite's interactions with exogenous fatty acids during the initial stages of infection., (Copyright © 2020 VanDussen et al.)

The intracellular protozoan parasite Toxoplasma gondii is capable of infecting most nucleated cells, where it survives in a specially modified compartment called the parasitophorous vacuole (PV). Interferon gamma (IFN-γ) is the major cytokine involved in activating cell-autonomous immune responses to inhibit parasite growth within this intracellular niche. In HeLa cells, IFN-γ treatment leads to ubiquitination of susceptible parasite strains, recruitment of the adaptors p62 and NDP52, and engulfment in microtubule-associated protein 1 light chain 3 (LC3)-positive membranes that restrict parasite growth. IFN-γ-mediated growth restriction depends on core members of the autophagy (ATG) pathway but not the initiation or degradative steps in the process. To explore the connection between these different pathways, we used permissive biotin ligation to identify proteins that interact with ATG5 in an IFN-γ-dependent fashion. Network analysis of the ATG5 interactome identified interferon-stimulated gene 15 (ISG15), which is highly upregulated by IFN treatment, as a hub connecting the ATG complex with other IFN-γ-induced genes, suggesting that it forms a functional link between the pathways. Deletion of ISG15 resulted in impaired recruitment of p62, NDP52, and LC3 to the PV and loss of IFN-γ-restricted parasite growth. The function of ISG15 required conjugation, and a number of ISGylated targets overlapped with the IFN-γ-dependent ATG5 interactome, including the adapter p62. Collectively, our findings establish a role for ISG15 in connecting the ATG pathway with IFN-γ-dependent restriction of T. gondii in human cells. IMPORTANCE Interferon(s) provide the primary defense against intracellular pathogens, a property ascribed to their ability to upregulate interferon-stimulated genes. Due to the sequestered niche occupied by Toxoplasma gondii , the host has elaborated intricate ways to target the parasite within its vacuole. One such mechanism is the recognition by a noncanonical autophagy pathway that envelops the parasite-containing vacuole and stunts growth in human cells. Remarkably, autophagy-dependent growth restriction requires interferon-γ, yet none of the classical components of autophagy are induced by interferon. Our studies draw a connection between these pathways by demonstrating that the antiviral protein ISG15, which is normally upregulated by interferons, links the autophagy-mediated control to ubiquitination of the vacuole. These findings suggest a similar link between interferon-γ signaling and autophagy that may underlie defense against other intracellular pathogens., (Copyright © 2020 Bhushan et al.)

Immunity to Toxoplasma gondii at early stages of infection in C57BL/6 mice depends on gamma interferon (IFN-γ) production by NK cells, while at later stages it is primarily mediated by CD8 T cells. We decided to explore the requirement for CD4 T cells during T. gondii infection in Batf3 -/- mice, which lack CD8α + dendritic cells (DCs) that are necessary for cross-presentation of cell-associated antigens to CD8 T cells. We show that in this immunodeficient background on a BALB/c background, CD4 T cells become important effector cells and are able to protect Batf3 -/- mice from infection with the avirulent strain RHΔ ku80 Δ rop5 Independently of the initial NK cell activation, CD4 T cells in wild-type and Batf3 -/- mice were the major source of IFN-γ. Importantly, memory CD4 T cells were sufficient to provide protective immunity following transfer into Batf3 -/- mice and secondary challenge with the virulent RHΔ ku80 strain. Collectively, these results show that under situations where CD8 cell responses are impaired, CD4 T cells provide an important alternative immune response to T. gondii IMPORTANCE Toxoplasma gondii is a widespread parasite of animals that causes zoonotic infections in humans. Although healthy individuals generally control the infection with only moderate symptoms, it causes serious illness in newborns and those with compromised immune systems such as HIV-infected AIDS patients. Because rodents are natural hosts for T. gondii , laboratory mice provide an excellent model for studying immune responses. Here, we used a combination of an attenuated mutant strain of the parasite that effectively vaccinates mice, with a defect in a transcriptional factor that impairs a critical subset of dendritic cells, to studying the immune response to infection. The findings reveal that in BALB/c mice, CD4 memory T cells play a dominant role in producing IFN-γ needed to control chronic infection. Hence, BALB/c mice may provide a more appropriate model for declining immunity seen in HIV-AIDS patients where loss of CD4 cells is associated with emergence of opportunistic infections., (Copyright © 2020 Tussiwand et al.)