The murine innate immune response against Toxoplasma gondii is predominated by the interaction of TLR11/12 with Toxoplasma profilin. However, mice lacking Tlr11 or humans, who do not have functional ...TLR11 or TLR12, still elicit a strong innate immune response upon Toxoplasma infection. The parasite factors that determine this immune response are largely unknown. Herein, we investigated two dense granule proteins (GRAs) secreted by Toxoplasma, GRA15 and GRA24, for their role in stimulating the innate immune response in Tlr11-/- mice and in human cells, which naturally lack TLR11/TLR12. Our results show that GRA15 and GRA24 synergistically shape the early immune response and parasite virulence in Tlr11-/- mice, with GRA15 as the predominant effector. Nevertheless, acute virulence in Tlr11-/- mice is still dominated by allelic combinations of ROP18 and ROP5, which are effectors that determine evasion of the immunity-related GTPases. In human macrophages, GRA15 and GRA24 play a major role in the induction of IL12, IL18 and IL1β secretion. We further show that GRA15/GRA24-mediated IL12, IL18 and IL1β secretion activates IFNγ secretion by peripheral blood mononuclear cells (PBMCs), which controls Toxoplasma proliferation. Taken together, our study demonstrates the important role of GRA15 and GRA24 in activating the innate immune response in hosts lacking TLR11.
The obligate intracellular parasite Toxoplasma gondii secretes effector proteins into the host cell that manipulate the immune response allowing it to establish a chronic infection. Crosses between ...the types I, II and III strains, which are prevalent in North America and Europe, have identified several secreted effectors that determine strain differences in mouse virulence. The polymorphic rhoptry protein kinase ROP18 was recently shown to determine the difference in virulence between type I and III strains by phosphorylating and inactivating the interferon-γ (IFNγ)-induced immunity-related GTPases (IRGs) that promote killing by disrupting the parasitophorous vacuole membrane (PVM) in murine cells. The polymorphic pseudokinase ROP5 determines strain differences in virulence through an unknown mechanism. Here we report that ROP18 can only inhibit accumulation of the IRGs on the PVM of strains that also express virulent ROP5 alleles. In contrast, specific ROP5 alleles can reduce IRG coating even in the absence of ROP18 expression and can directly interact with one or more IRGs. We further show that the allelic combination of ROP18 and ROP5 also determines IRG evasion and virulence of strains belonging to other lineages besides types I, II and III. However, neither ROP18 nor ROP5 markedly affect survival in IFNγ-activated human cells, which lack the multitude of IRGs present in murine cells. These findings suggest that ROP18 and ROP5 have specifically evolved to block the IRGs and are unlikely to have effects in species that do not have the IRG system, such as humans.
Toxoplasma gondii is a parasite that replicates within a specialized compartment called the parasitophorous vacuole (PV), which is surrounded by the PV membrane (PVM). To obtain essential nutrients, ...Toxoplasma must transport molecules across the PVM, a process mediated by the secreted parasite proteins GRA17 and GRA23. These proteins form pores in the PVM through which small molecules can diffuse in and out of the PV. GRA17 and GRA23 are synthetically lethal, suggesting that at least one pore type is essential for parasite survival. In the 'nutrient sensitized' Δgra17 strain it is likely that other Toxoplasma genes become essential, because they mediate nutrient acquisition from the host or are involved in the trafficking of GRA23 to the PVM. To identify these genes, a genome-wide loss-of-function screen was performed in wild-type and Δgra17 parasites, which identified multiple genes that were synthetically sick/lethal with GRA17. Several of these genes were involved in the correct localization of GRAs, including GRA17/GRA23, to the PVM. One of the top hits, GRA72, was predicted to form a pore on the PVM, and its deletion led to the formation of enlarged "bubble vacuoles" with reduced PVM small molecule permeability, similar to what was previously observed for Δgra17 parasites. Furthermore, Δgra72 parasites had reduced in vitro growth and virulence in mice. These findings suggest that in the absence of GRA17, other genes become essential, likely because they play a role in the proper localization of GRA23 (and other GRAs) or because they determine host-derived nutrient acquisition at the PVM.
Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa, has the unusual ability to infect virtually any warm-blooded animal. It is an extraordinarily successful parasite, ...infecting an estimated 30% of humans worldwide. The outcome of Toxoplasma infection is highly dependent on allelic differences in the large number of effectors that the parasite secretes into the host cell. Here, we show that the largest determinant of the virulence difference between two of the most common strains of Toxoplasma is the ROP5 locus. This is an unusual segment of the Toxoplasma genome consisting of a family of 4-10 tandem, highly divergent genes encoding pseudokinases that are injected directly into host cells. Given their hypothesized catalytic inactivity, it is striking that deletion of the ROP5 cluster in a highly virulent strain caused a complete loss of virulence, showing that ROP5 proteins are, in fact, indispensable for Toxoplasma to cause disease in mice. We find that copy number at this locus varies among the three major Toxoplasma lineages and that extensive polymorphism is clustered into hotspots within the ROP5 pseudokinase domain. We propose that the ROP5 locus represents an unusual evolutionary strategy for sampling of sequence space in which the gene encoding an important enzyme has been (i) catalytically inactivated, (ii) expanded in number, and (iii) subject to strong positive selection. Such a strategy likely contributes to Toxoplasma's successful adaptation to a wide host range and has resulted in dramatic differences in virulence.
About the Authors: Shruthi Krishnamurthy Affiliation: Department of Pathology, Microbiology and Immunology, University of California Davis, Davis, California, United States of America ORCID ...http://orcid.org/0000-0003-4540-6613 Eleni K. Konstantinou Affiliation: Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States of America ORCID http://orcid.org/0000-0002-7155-5770 Lucy H. Young Affiliation: Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States of America ORCID http://orcid.org/0000-0001-8634-7512 Daniel A. Gold Affiliation: Department of Pathology, Microbiology and Immunology, University of California Davis, Davis, California, United States of America Jeroen P. J. Saeij * E-mail: jsaeij@ucdavis.edu Affiliation: Department of Pathology, Microbiology and Immunology, University of California Davis, Davis, California, United States of America ORCID http://orcid.org/0000-0003-0289-7109Citation: Krishnamurthy S, Konstantinou EK, Young LH, Gold DA, Saeij JPJ (2017) The human immune response to Toxoplasma: Autophagy versus cell death. Humans also lack the multitude of murine immunity-related GTPases (IRGs) that are induced upon IFNgamma stimulation and play a crucial role in the destruction of the membrane surrounding the parasitophorous vacuole (PV) in which Toxoplasma resides in the host cytoplasm. ...the mechanisms involved in the production of IFNgamma in humans (Fig 1) differ from those in mice and the pathways that mediate parasite clearance are less well understood (reviewed in 1). See the main text for explanations. https://doi.org/10.1371/journal.ppat.1006176.g001 IFNgamma-dependent, noncanonical autophagy-mediated clearance of Toxoplasma in humans Autophagy is a degradation process that clears cytoplasmic material such as misfolded proteins and damaged organelles. During autophagy, cytosolic microtubule-associated protein light chain 3 (LC3), a ubiquitin-like protein, is processed and conjugated to the lipid phosphatidylethanolamine (PE)...
Toxoplasma gondii is a protozoan pathogen in the phylum Apicomplexa that resides within an intracellular parasitophorous vacuole (PV) that is selectively permeable to small molecules through ...unidentified mechanisms. We have identified GRA17 as a Toxoplasma-secreted protein that localizes to the parasitophorous vacuole membrane (PVM) and mediates passive transport of small molecules across the PVM. GRA17 is related to the putative Plasmodium translocon protein EXP2 and conserved across PV-residing Apicomplexa. The PVs of GRA17-deficient parasites have aberrant morphology, reduced permeability to small molecules, and structural instability. GRA17-deficient parasites proliferate slowly and are avirulent in mice. These GRA17-deficient phenotypes are rescued by complementation with Plasmodium EXP2. GRA17 functions synergistically with a related protein, GRA23. Exogenous expression of GRA17 or GRA23 alters the membrane conductance properties of Xenopus oocytes in a manner consistent with a large non-selective pore. Thus, GRA17 and GRA23 provide a molecular basis for PVM permeability and nutrient access.
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•GRA17 and GRA23 are secreted proteins unique to PV-residing apicomplexans•GRA17 affects the transfer of small molecules through the PV and in vivo virulence•The GRA17 loss-of-function phenotypes can be rescued by Plasmodium EXP2•GRA17 or GRA23 expression alters the membrane conductance of Xenopus oocytes
Toxoplasma gondii resides within an intracellular parasitophorous vacuole (PV) selectively permeable to small molecules. Gold et al. identify GRA17 and GRA23 as secreted Toxoplasma proteins that mediate the passage of small molecules between the host cytoplasm and PV, providing a molecular explanation for how these vacuole-residing parasites access host nutrients.
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.
is an exceptionally successful parasite that infects a very broad host range, including humans, across the globe. The outcome of infection differs remarkably between hosts, ranging from acute death ...to sterile infection. These differential disease patterns are strongly influenced by both host- and parasite-specific genetic factors. In this review, we discuss how the clinical outcome of toxoplasmosis varies between hosts and the role of different immune genes and parasite virulence factors, with a special emphasis on
induced ileitis and encephalitis.
Apicomplexan parasites are leading causes of human and livestock diseases such as malaria and toxoplasmosis, yet most of their genes remain uncharacterized. Here, we present the first genome-wide ...genetic screen of an apicomplexan. We adapted CRISPR/Cas9 to assess the contribution of each gene from the parasite Toxoplasma gondii during infection of human fibroblasts. Our analysis defines ∼200 previously uncharacterized, fitness-conferring genes unique to the phylum, from which 16 were investigated, revealing essential functions during infection of human cells. Secondary screens identify as an invasion factor the claudin-like apicomplexan microneme protein (CLAMP), which resembles mammalian tight-junction proteins and localizes to secretory organelles, making it critical to the initiation of infection. CLAMP is present throughout sequenced apicomplexan genomes and is essential during the asexual stages of the malaria parasite Plasmodium falciparum. These results provide broad-based functional information on T. gondii genes and will facilitate future approaches to expand the horizon of antiparasitic interventions.
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•CRISPR enables the first genome-wide loss-of-function screen in T. gondii•The screen measures each gene’s impact during infection of human fibroblasts•Functional assays validate the essentiality of formerly uncharacterized proteins•An invasion factor found in all apicomplexans is essential in malarial parasites
The first genome-wide genetic screen of an apicomplexan parasite identifies an invasion factor essential for the fitness of all parasites in this phylum, including those causing malaria and toxoplasmosis.
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