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.

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.

c Toxoplasma gondii transmission 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 the Toxoplasma secreted effectors rhoptry 16 kinase (ROP16) and dense granule 15 (GRA15) activate the JAK-STAT3/6 and NF-B signaling pathways, respectively, we explored whether a particular combination of these effectors impacted intestinal inflammation and parasite survival in 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 I strain 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 I suggested 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 in Toxoplasma-infected cells. In summary, only the combined expression of both GRA15 and ROP16 promoted host resistance to acute oral infection, and Toxoplasma may possibly target the STAT5 signaling pathway to generate protective immunity in the gut.

Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identify 353 Toxoplasma genes 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 in Toxoplasma and provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγ-activated macrophages.