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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. Using a genome-wide CRISPR screen, the authors here identify Toxoplasma genes that counteract the anti-parasitic activity of interferon gamma in murine macrophages. One of these genes, dense granule protein GRA45, is critical for the trafficking of GRA effectors and affects virulence in mice.

Toxoplasma gondii , a zoonotic apicomplexan that infects over a billion people worldwide, can cause early death in immunocompromised individuals and defects in foetal brain development. Toxoplasma is also a major cause of abortion in small ruminants. When Toxoplasma encounters host cells, several outcomes are possible. For example, the parasite can enter the host cell or can inject its effector proteins into the cell without entering. These heterogenous outcomes occur simultaneously in the same host and likely determine disease pathogenesis. Yet, current knowledge of host- Toxoplasma interactions is largely based on averaged responses in bulk cell populations. Here, we employed single cell RNA (scRNA) and bulk RNA sequencing to investigate the transcriptional profiles that underpin heterogenous host- Toxoplasma interaction in human peripheral blood mononuclear cells. We observed that Toxoplasma preferentially infects and elicits transcriptional responses in dendritic cells in human blood. Additionally, we observed that monocytes adopt a dendritic cell-like transcriptional profile over the course of infection. Using genes expressed in sorted host cell populations representative of the different heterogenous host- Toxoplasma interaction outcomes as a reference panel, we show that genes expressed in cells infected via phagocytosis are largely expressed in dendritic cells. Thus, by integrating scRNA and bulk RNA sequencing, our study unveils the transcriptional profiles of diverse Toxoplasma -host cell interaction outcomes, providing novel avenues for targeted investigations into host gene functions during Toxoplasma infections.

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.

The obligate intracellular parasite, , is highly prevalent among livestock species. Although cattle are generally resistant to strains circulating in Europe and North America, the underlying mechanisms are largely unknown. Here, we report that bovine bone marrow-derived macrophage (BMDM) pre-stimulated with interferon gamma (IFNγ) restricts intracellular growth independently of nitric oxide. While promoted the expression of genes associated with alternative macrophage activation and lipid metabolism, IFNγ abrogated parasite-induced transcriptional responses and promoted the expression of genes linked to the classical macrophage activation phenotype. Additionally, several chemokines, including , that are linked to parasite-induced activation of the /β-catenin signaling were highly expressed in -exposed naïve BMDMs. A chemical /β-catenin signaling pathway antagonist (IWR-1-endo) significantly reduced intracellular parasite burden in naïve BMDMs, suggesting that activates this pathway to evade bovine macrophage anti-parasitic responses. Congruently, intracellular burden of a mutant strain (RHΔ ) that does not secrete dense granule proteins into the host cell, which is an essential requirement for parasite-induced activation of the /β-catenin pathway, was significantly reduced in naïve BMDMs. However, both the /β-catenin antagonist and RH Δ5 did not abolish parasite burden differences in naïve and IFNγ-stimulated BMDMs. Finally, we observed that parasites infecting IFNγ-stimulated BMDMs largely express genes associated with the slow dividing bradyzoite stage. Overall, this study provides novel insights into bovine macrophage transcriptional response to . It establishes a foundation for a mechanistic analysis IFNγ-induced bovine anti- responses and the counteracting survival strategies.

Background Infectious Salmon Anaemia Virus (ISAV) is an Orthomixovirus that represents a large problem for salmonid aquaculture worldwide. Current prevention and treatment methods are only partially effective. Genetic selection and genome engineering have the potential to develop ISAV resistant salmon stocks. Both strategies can benefit from an improved understanding of the genomic regulation of ISAV pathogenesis. Here, we used single-cell RNA sequencing of an Atlantic salmon cell line to provide the first high dimensional insight into the transcriptional landscape that underpins host-virus interaction during early ISAV infection. Results Salmon head kidney (SHK-1) cells were single-cell RNA sequenced at 24, 48 and 96 h post-ISAV challenge. At 24 h post infection, cells showed expression signatures consistent with viral entry, with genes such as PI3K, FAK or JNK being upregulated relative to uninfected cells. At 48 and 96 h, infected cells showed a clear anti-viral response, characterised by the expression of IFNA2 or IRF2. Uninfected bystander cells at 48 and 96 h also showed clear transcriptional differences, potentially suggesting paracrine signalling from infected cells. These bystander cells expressed pathways such as mRNA sensing, RNA degradation, ubiquitination or proteasome; and up-regulation of mitochondrial ribosome genes also seemed to play a role in the host response to the infection. Correlation between viral and host genes revealed novel genes potentially key for this fish-virus interaction. Conclusions This study has increased our understanding of the cellular response of Atlantic salmon during ISAV infection and revealed host-virus interactions at the cellular level. Our results highlight various potential key genes in this host-virus interaction, which can be manipulated in future functional studies to increase the resistance of Atlantic salmon to ISAV.

Background Differential isoform usage is an important driver of inter-individual phenotypic diversity and is linked to various diseases and traits. However, accurately detecting the differential usage of different gene transcripts between groups can be difficult, in particular in less well annotated genomes where the spectrum of transcript isoforms is largely unknown. Results We investigated whether machine learning approaches can detect differential isoform usage based purely on the distribution of reads across a gene region. We illustrate that gradient boosting and elastic net approaches can successfully identify large numbers of genes showing potential differential isoform usage between Europeans and Africans, that are enriched among relevant biological pathways and significantly overlap those identified by previous approaches. We demonstrate that diversity at the 3′ and 5′ ends of genes are primary drivers of these differences between populations. Conclusion Machine learning methods can effectively detect differential isoform usage from read fraction data, and can provide novel insights into the biological differences between groups.

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. Upon invasion of Lewis rat macrophages, Toxoplasma rapidly induces programmed cell death (pyroptosis), which prevents Toxoplasma replication, possibly explaining the resistance of the Lewis rat to Toxoplasma . Using a chemical mutagenesis screen, we identified Toxoplasma mutants that no longer induced pyroptosis. Whole-genome sequencing led to the identification of three Toxoplasma parasitophorous vacuole-localized dense granule proteins, GRA35, GRA42, and GRA43, that are individually required for induction of Lewis rat macrophage pyroptosis. Macrophage infection with Δ gra35 , Δ gra42 , and Δ gra43 parasites led to greatly reduced cell death rates and enhanced parasite replication. Lewis rat macrophages infected with parasites containing a single, double, or triple deletion of these GRAs showed similar levels of cell viability, suggesting that the three GRAs function in the same pathway. Deletion of GRA42 or GRA43 resulted in GRA35 (and other GRAs) being retained inside the parasitophorous vacuole instead of being localized to the parasitophorous vacuole membrane. Despite having greatly enhanced replication in Lewis rat macrophages in vitro , Δ gra35 , Δ gra42 , and Δ gra43 parasites did not establish a chronic infection in Lewis rats. Toxoplasma did not induce F344 rat macrophage pyroptosis, but F344 rats infected with Δ gra35 , Δ gra42 , and Δ gra43 parasites had reduced cyst numbers. Thus, these GRAs determined parasite in vivo fitness in F344 rats. Overall, our data suggest that these three Toxoplasma dense granule proteins play a critical role in establishing a chronic infection in vivo , independently of their role in mediating macrophage pyroptosis, likely due to their importance in regulating protein localization to the parasitophorous vacuole membrane. IMPORTANCE 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.

Autologous administration of attenuated Theileria parva -infected cells induces immunity to T. parva in cattle. The mechanism of attenuation, however, is largely unknown. Here, we used RNA sequencing of pathogenic and attenuated T. parva -infected T-cells to elucidate the transcriptional changes underpinning attenuation. We observed differential expression of several host genes, including TRAIL , PD-1 , TGF-β and granzymes that are known to regulate inflammation and proliferation of infected cells. Importantly, many genes linked with the attenuation of the related T. annulata -infected cells were not dysregulated in this study. Furthermore, known T. parva antigens were not dysregulated in attenuated relative to pathogenic cells, indicating that attenuation is not due to enhanced immunogenicity. Overall this study suggests that attenuation is driven by a decrease in proliferation and restoration of the inflammatory profile of T. parva -infected cells. Additionally, it provides a foundation for future mechanistic studies of the attenuation phenotype in Theileria -infected cells.

is an apicomplexan parasite responsible for bovine neosporosis, a major cause of abortion in cattle worldwide. rhoptry protein 2 (NcROP2) has been identified as an essential factor in host cell invasion and parasitophorous vacuole formation, making it a potential target for disease control strategies. In this study, we generated knockout ( ) mutants using CRISPR/Cas9 technology to assess their role in parasite virulence. In a pregnant mouse model, parasites exhibited reduced virulence, as indicated by increased neonatal survival rates and lower parasite burden in the brain and attenuated clinical signs in the dams compared to the wild-type (Nc-Spain7) parental strain. Additionally, the mutants exhibited impaired proliferation and significantly induced the expression of interferon-stimulated genes in bovine monocyte-derived macrophages infected for 60 hours. Transcriptomic analysis further revealed a shift in parasite gene expression, with an upregulation of stress-related and bradyzoite markers. Functional assays confirmed that Δ parasites were less susceptible to IFN-γ-mediated inhibition and displayed an enhanced ability to convert to the semi-dormant bradyzoite stage. These findings highlight NcROP2 as a key virulence factor involved in immune evasion and parasite proliferation, providing new insights into infection pathogenesis and potential avenues for vaccine development.

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.