Toxoplasma gondii is an intracellular parasite that infects a wide range of warm-blooded species. Rats vary in their susceptibility to this parasite. The Toxo1 locus conferring Toxoplasma resistance in rats was previously mapped to a region of chromosome 10 containing Nlrp1. This gene encodes an inflammasome sensor controlling macrophage sensitivity to anthrax lethal toxin (LT) induced rapid cell death (pyroptosis). We show here that rat strain differences in Toxoplasma infected macrophage sensitivity to pyroptosis, IL-1β/IL-18 processing, and inhibition of parasite proliferation are perfectly correlated with NLRP1 sequence, while inversely correlated with sensitivity to anthrax LT-induced cell death. Using recombinant inbred rats, SNP analyses and whole transcriptome gene expression studies, we narrowed the candidate genes for control of Toxoplasma-mediated rat macrophage pyroptosis to four genes, one of which was Nlrp1. Knockdown of Nlrp1 in pyroptosis-sensitive macrophages resulted in higher parasite replication and protection from cell death. Reciprocally, overexpression of the NLRP1 variant from Toxoplasma-sensitive macrophages in pyroptosis-resistant cells led to sensitization of these resistant macrophages. Our findings reveal Toxoplasma as a novel activator of the NLRP1 inflammasome in rat macrophages. Inflammasomes are multiprotein complexes that are a major component of the innate immune system. They contain “sensor” proteins that are responsible for detecting various microbial and environmental danger signals and function by activating caspase-1, an enzyme that mediates cleavage and release of the pro-inflammatory cytokines, IL-1β and IL-18. Toxoplasma gondii is a highly successful protozoan parasite capable of infecting a wide range of host species that have variable levels of resistance. Rat strains have been previously shown to vary in their susceptibility to this parasite. We report here that rat macrophages from different inbred strains also vary in sensitivity to Toxoplasma induced lysis. We find that NLRP1, an inflammasome sensor whose only known agonist is anthrax LT, is also activated by Toxoplasma infection. In rats there is a perfect correlation between NLRP1 sequence and macrophage sensitivity to Toxoplasma-induced rapid cell death, inhibition of parasite proliferation, and IL-1β/IL-18 processing. Nlrp1 genes from sensitive rat macrophages can confer sensitivity to this rapid cell death when expressed in Toxoplasma resistant rat macrophages. Our findings suggest Toxoplasma is a new activator of the NLRP1 inflammasome.

Most isolates of Toxoplasma from Europe and North America fall into one of three genetically distinct clonal lineages, the type I, II and III lineages. However, in South America these strains are rarely isolated and instead a great variety of other strains are found. T. gondii strains differ widely in a number of phenotypes in mice, such as virulence, persistence, oral infectivity, migratory capacity, induction of cytokine expression and modulation of host gene expression. The outcome of toxoplasmosis in patients is also variable and we hypothesize that, besides host and environmental factors, the genotype of the parasite strain plays a major role. The molecular basis for these differences in pathogenesis, especially in strains other than the clonal lineages, remains largely unexplored. Macrophages play an essential role in the early immune response against T. gondii and are also the cell type preferentially infected in vivo. To determine if non-canonical Toxoplasma strains have unique interactions with the host cell, we infected murine macrophages with 29 different Toxoplasma strains, representing global diversity, and used RNA-sequencing to determine host and parasite transcriptomes. We identified large differences between strains in the expression level of known parasite effectors and large chromosomal structural variation in some strains. We also identified novel strain-specifically regulated host pathways, including the regulation of the type I interferon response by some atypical strains. IFNβ production by infected cells was associated with parasite killing, independent of interferon gamma activation, and dependent on endosomal Toll-like receptors in macrophages and the cytoplasmic receptor retinoic acid-inducible gene 1 (RIG-I) in fibroblasts. Toxoplasmosis is caused by the protozoan parasite Toxoplasma gondii. The parasite is found throughout the world. When humans are infected, few have symptoms because a healthy immune system usually prevents the parasite from causing illness. Nevertheless, cases of severe disease in otherwise healthy individuals have been observed. These cases are usually a result of infection with less common atypical strains of Toxoplasma. Factors associated with virulence in the atypical strains are not well understood. Here, we infected host cells with 29 different strains of Toxoplasma, and performed high-throughput RNA sequencing of both host cells and parasites. We found significant differences in gene expression profiles between strains. Host cell transcriptional response also varied substantially depending on the infecting strain. Specifically, we found that a small group of atypical strains are able to induce production of type I interferons, which are immunomodulatory cytokines. Interferon production is a result of the elimination of internalized parasites through a novel killing mechanism. The dataset we generated is a valuable tool for identification of host cell targets of Toxoplasma secreted effectors and can contribute to our understanding of why certain Toxoplasma strains are more prone to cause severe disease in humans.

Toxoplasma gondiitransmission between intermediate hosts is dependent on the ingestion of walled cysts formed during the chronic phase of infection. Immediately following consumption, the parasite must ensure survival of the host by preventing adverse inflammatory responses and/or by limiting its own replication. Since theToxoplasmasecreted effectors rhoptry 16 kinase (ROP16) and dense granule 15 (GRA15) activate the JAK-STAT3/6 and NF-κB signaling pathways, respectively, we explored whether a particular combination of these effectors impacted intestinal inflammation and parasite survivalin vivo. Here we report that expression of the STAT-activating version of ROP16 in the type II strain (strain II+ROP16_I) promotes host resistance to oral infection only in the context of endogenous GRA15 expression. Protection was characterized by a lower intestinal parasite burden and dampened inflammation. Host resistance to the II+ROP16_Istrain occurred independently of STAT6 and the T cell coinhibitory receptors B7-DC and B7-H1, two receptors that are upregulated by ROP16. In addition, coexpression of ROP16 and GRA15 enhanced parasite susceptibility within tumor necrosis factor alpha/gamma interferon-stimulated macrophages in a STAT3/6-independent manner. Transcriptional profiling of infected STAT3- and STAT6-deficient macrophages and parasitized Peyer's patches from mice orally challenged with strain II+ROP16_Isuggested that ROP16 activated STAT5 to modulate host gene expression. Consistent with this supposition, the ROP16 kinase induced the sustained phosphorylation and nuclear localization of STAT5 inToxoplasma-infected cells. In summary, only the combined expression of both GRA15 and ROP16 promoted host resistance to acute oral infection, andToxoplasmamay possibly target the STAT5 signaling pathway to generate protective immunity in the gut.

Macrophages play an essential role in the early immune response againstToxoplasmaand are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasmaactivities in macrophages. Using a genome-wide CRISPR screen we identify 353Toxoplasmagenes that determine parasite fitness in naїve or IFNγ-activated murine macrophages, seven of which are further confirmed. We show that one of these genes encodes dense granule protein GRA45, which has a chaperone-like domain, is critical for correct localization of GRAs into the PVM and secretion of GRA effectors into the host cytoplasm. Parasites lacking GRA45 are more susceptible to IFNγ-mediated growth inhibition and have reduced virulence in mice. Together, we identify and characterize an important chaperone-like GRA inToxoplasmaand provide a resource for the community to further explore the function ofToxoplasmagenes that determine fitness in IFNγ-activated macrophages.

Inflammasomes are major components of the innate immune system and are responsible for detecting various microbial and environmental danger signals. Upon invasion of Lewis rat macrophages, the parasite rapidly activates the NLRP1 inflammasome, resulting in pyroptosis and elimination of the parasite’s replication niche. The work reported here revealed that Toxoplasma GRA35, GRA42, and GRA43 are required for induction of Lewis rat macrophage pyroptosis. GRA42 and GRA43 mediate the correct localization of other GRAs, including GRA35, to the parasitophorous vacuole membrane. These three GRAs were also found to be important for parasite in vivo fitness in a Toxoplasma -susceptible rat strain, independently of their role in NLRP1 inflammasome activation, suggesting that they perform other important functions. Thus, this study identified three GRAs that mediate the induction of Lewis rat macrophage pyroptosis and are required for pathogenesis of the parasite.

Background: Accurate gene model predictions and annotation of alternative splicing events are imperative for genomic studies in organisms that contain genes with multiple exons. Currently most gene models for the intracellular parasite,Toxoplasma gondii, are based on computer model predictions without cDNA sequence verification. Additionally, the nature and extent of alternative splicing inToxoplasma gondiiis unknown. In this study, we usedde novotranscript assembly and the published type II (ME49) genomic sequence to quantify the extent of alternative splicing inToxoplasmaand to improve the currentToxoplasmagene annotations.Results: We used high-throughput RNA-sequencing data to assemble full-length transcripts, independently of a reference genome, followed by gene annotation based on the ME49 genome. We assembled 13,533 transcripts overlapping with known ME49 genes in ToxoDB and then used this set to; a) improve the annotation in the untranslated regions of ToxoDB genes, b) identify novel exons within protein-coding ToxoDB genes, and c) report on 50 previously unidentified alternatively spliced transcripts. Additionally, we assembled a set of 2,930 transcripts not overlapping with any known ME49 genes in ToxoDB. From this set, we have identified 118 new ME49 genes, 18 novelToxoplasmagenes, and putative non-coding RNAs.Conclusion: RNA-seq data andde novotranscript assembly provide a robust way to update incompletely annotated genomes, like theToxoplasmagenome. We have used RNA-seq to improve the annotation of severalToxoplasmagenes, identify alternatively spliced genes, novel genes, novel exons, and putative non-coding RNAs.