Toxoplasma Research

Toxoplasma proteins that modulate the immune response

The majority of Toxoplasma isolates from Europe and North America belong to three distinct clonal lines, referred to as types I, II and III. The three types have been shown to differ widely in a number of phenotypes in mice such as virulence, persistence, migratory capacity, attraction of different cell-types and induction of cytokine expression. Recent data suggest that such differences may also exist in human infection. In South America many other Toxoplasma strains exist, some of which can cause severe disease even in healthy individuals. One of our long-term goals is to understand how distinct Toxoplasma strains differ in their ability to cause disease in humans. Determining how particular Toxoplasma genotypes differ in their capacity to induce pathology in a particular animal species could enable prediction of the outcome of infection based on the genotype of the infecting organism. For example, not all seropositive AIDS patients develop toxoplasmic encephalitis; the ones that do might be infected with a particular subset of parasite strains. Similarly, seroconversion during pregnancy does not always lead to infection of the fetus; this might be a result of variability in the ability of different strains to cross the placental barrier.

Toxoplasma can cause disease because of tissue damage associated with high parasite burdens. It can also stimulate a hyperactive immune response that can cause inflammation and tissue damage. The Toxoplasma proteins that are secreted into the host cell are called ROPs and GRAs, which are secreted from Toxoplasma secretory organelles called rhoptries and dense granules. Toxoplasma has a haploid genome with 14 chromosomes that total 65 Mbp in size, representing ~8000 genes ( Classical genetic crosses can be performed in Toxoplasma and several experimental crosses have been used to generate genetic linkage maps. To identify the Toxoplasma loci involved in virulence, virulence was mapped in F1 progeny derived from crosses between type II and type III strains and five virulence loci were identified. We, and others, established that Toxoplasma virulence caused by high parasite burdens in mice, is largely determined by two proteins that cooperatively block mouse innate immune mechanisms that would otherwise destroy the vacuole in which the parasite lives. These proteins, ROP18 (a Toxoplasma-secreted kinase) and ROP5 (a family of secreted pseudokinases), cooperatively block the murine interferon-gamma (IFN)γ-induced immunity related GTPases (IRGs), thereby preventing destruction of the vacuole in which the parasite lives. The expansion and subsequent diversification of Toxoplasma effector genes, such as ROP5, is likely an evolutionary strategy of multi-host parasites to be able to bind to the divergent substrates in different host species, for example immunity related GTPases in mice and rats. We are investigating this co-evolution of ROP5 with these GTPases from different mouse and rat strains with the goal of linking host differences in susceptibility to specific Toxoplasma strains with specific ROP5-GTPase combinations.

($Denotes shared first author. *Denotes graduate students from my lab. #Denotes Co-corresponding authors)
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  • Lim D, Gold DA, Julien L, Rosowski EE*, Niedelman W*, Yaffe MB#, Saeij JP#. Structure of the Toxoplasma gondii ROP18 kinase domain reveals a second ligand binding pocket required for acute virulence. The Journal of Biological Chemistry. 288(48):34968-80; 2013.
  • Niedelman W*, Gold DA, Rosowski EE*, Sprokholt J, Lim D, Farid A, Melo MB, Spooner E, Yaffe MB, Saeij JP. The rhoptry proteins ROP18 and ROP5 mediate Toxoplasma gondii evasion of the murine, but not the human, interferon-gamma response. PLoS Pathogens. 8(6):e1002784; 2012.
  • Lim DC, Cooke BM, Doerig C, Saeij JP. Toxoplasma and Plasmodium protein kinases: roles in invasion and host cell remodelling. International Journal for Parasitology. 42(1):21-32;2012. [Review article]
  • Virreira-Winter S, Niedelman W*, Jensen KD, Rosowski EE*, Julien L, Spooner E, Caradonna K, Burleigh BA, Saeij JP, Ploegh HL, Frickel E. Determinants of GBP recruitment to Toxoplasma gondii vacuoles and the parasitic factors that control it. PLoS One. 6(9):e24434; 2011.
  • Reese ML, Zeiner GM, Saeij JP, Boothroyd JC, Boyle JP. Polymorphic family of injected pseudokinases is paramount in Toxoplasma virulence. PNAS 108(23):9625-30; 2011.
  • Saeij JP$, Boyle JP$, Coller SC, Taylor S, Sibley LD, Brooke-Powell ET, Ajioka JW, Boothroyd JC. Polymorphic secreted kinases are key virulence factors in toxoplasmosis. Science 314(5806):1780-1783; 2006.
  • Saeij JP$, Boyle JP$, Grigg ME, Arrizabalaga G, Boothroyd JC. Bioluminescence imaging of Toxoplasma gondii infection in living mice reveals dramatic differences between strains. Infection and Immunity. 73(2): 695-702; 2005.
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Toxoplasma proteins that modulate host signaling pathways

Different Toxoplasma strains also differ significantly in the modulation of host signaling pathways. We have infected macrophages with 29 different Toxoplasma strains, representing global diversity, and determined the macrophage and parasite transcriptomes. This has provided global insight into host cell and parasite transcriptional responses upon infection and allowed identification of novel strain specific host cell responses and parasite effectors. Overall, we have shown that two Toxoplasma proteins play a major role in the modulation of macrophage function and thereby determine the level of immune-induced inflammation. These proteins are ROP16 (a secreted kinase) and GRA15 (a secreted protein with no characterized motifs). ROP16 activates the signal transducer and activator of transcription (STAT)3 and STAT6 transcription factors resulting in the repression of inflammation. GRA15 activates NF-kB, a transcription factor involved in the activation of the inflammatory response. We are now combining genetic and biochemical approaches to identify host factors that interact with GRA15 and to identify the host genes and pathways that mediate ROP16-induced anti-inflammatory effects. We are also identifying and characterizing novel ROPs and GRAs and investigating their role in the modulation of the host cell. ROPs and GRAs are often present in slightly different versions in different strains and the exact combination of these protein versions determine if a particular Toxoplasma strain is more or less virulent or causes more or less inflammation.
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  • Melo MB, Nguyen QP, Cordeiro C, Hassan MA, Yang N*, McKell R*, Rosowski EE*, Julien L, Butty V, Darde M-L, Ajzenberg D, Fitzgerald K. Young LH, Saeij JP. Transcriptional analysis of murine macrophages infected with different Toxoplasma strains identifies novel regulation of host signaling pathways. PLoS Pathogens. 9(12):e1003809; 2013.
  • Camejo A, Gold DA, Lu D*, McFetridge K, Julien L, Yang N*, Jensen KD, Saeij JP. Identification of three novel Toxoplasma gondii rhoptry proteins. International Journal for Parasitology. S0020-7519(13)00222-1; 2013. [Epub ahead of print].
  • Yang N*, Farrell A, Niedelman W*, Melo MB, Lu D*, Julien L, Marth GT, Gubbels MJ, Saeij JP. Genetic basis for phenotypic differences between different Toxoplasma gondii type I strains. BMC Genomics. 14:467; 2013.
  • Jensen KD, Hu K, Whitmarsh RJ, Hassan MA, Julien L, Lu D*, Chen L, Hunter CA, Saeij JP. Toxoplasma gondii rhoptry 16 kinase promotes host resistance to oral infection and intestinal inflammation only in the context of the dense granule protein GRA15. Infection and Immunity. 81(6):2156-67; 2013.
  • Hassan MA, Melo MB, Haas B, Jensen KD, Saeij JP. De novo reconstruction of the Toxoplasma gondii transcriptome improves on the current genome annotation and reveals alternatively spliced transcripts and putative long non-coding RNAs. BMC Genomics. 13(1):696; 2012.
  • Jensen KD, Wang Y, Tait ED, Shastri AJ, Hu K, Cornel L, Boedec E, Ong Y, Chien Y, Hunter CA, Boothroyd JC, Saeij JP. Toxoplasma polymorphic effectors determine macrophage polarization and intestinal inflammation. Cell Host & Microbe 9(6):472-83; 2011.
  • Melo MB$, Jensen KD$, Saeij JP. Toxoplasma gondii effectors are master regulators of the inflammatory response. Trends in Parasitology. 27(11):487-95; 2011.
  • Rosowski EE$*, Lu D$*, Julien L, Rodda L, Gaiser R, Jensen KD, Saeij JP. Strain-specific activation of the NF-κB pathway by GRA15, a novel Toxoplasma dense granule protein. Journal of Experimental Medicine 208(1):195-212; 2011.
  • Blader IJ, Saeij JPJ. Communication between Toxoplasma gondii and its host: impact on parasitegrowth, development, immune evasion, and virulence. APMIS (acta pathologica, microbiologica, et immunologica Scandinavica). 117(5-6):458-76; 2009.
  • Saeij JP$, Coller S$, Boyle JP, Jerome ME, White MW, Boothroyd JC. Toxoplasma co-opts host gene expression by injection of a polymorphic kinase homologue. Nature 445(7125): 324-327; 2007.
  • Saeij JP, Boyle JP, Boothroyd JC. Differences among the three major strains of Toxoplasma gondii and their specific interactions with the infected host. Trends in Parasitology. 21(10): 476-81; 2005.
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Toxoplasma proteins that determine virulence in humans

Humans do not have the wide diversity of immunity related GTPases that many rodents have and ROP5 and ROP18 do not seem to play a role in blocking IFNγ-induced toxoplasmacidal mechanisms in human cells. We recently determined that Toxoplasma infection induces cell death of interferon-gamma stimulated human fibroblasts, which induces early parasite egress and limits parasite replication. We are currently determining the host and parasite proteins involved.
The IFNγ response, mediated by the STAT1 transcription factor, is crucial for host defense against Toxoplasma, but prior infection with Toxoplasma can inhibit this response, which is thought to allow the parasite to establish a chronic infection. We determined that Toxoplasma inhibits the dissociation of STAT1 from DNA, preventing its recycling and further rounds of STAT1-mediated transcriptional activation. We are using a biochemical approach to identify the Toxoplasma protein that mediates the inhibition of the STAT1 transcriptional response.
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  • Rosowski EE*, Nguyen QP, Camejo A, Spooner E, Saeij JP. Toxoplasma gondii inhibits Gamma Interferon (IFN-γ)- and IFN-β-induced host cell STAT1 transcriptional activity by increasing the association of STAT1 with DNA. Infection and Immunity. 82(2):706-719; 2014.
  • Niedelman W*, Sprokholt JK, Clough B, Frickel EM, Saeij JP. Cell death of interferon-gamma stimulated human fibroblasts upon Toxoplasma gondii infection induces early parasite egress and limits parasite replication. Infection and Immunity. 81(12):4341-9; 2013.
  • Rosowski EE*, Saeij JP. Toxoplasma gondii clonal strains all inhibit STAT1 transcriptional activity but polymorphic effectors differentially modulate IFNγ induced gene expression and STAT1 phosphorylation. PLoS One. 7(12):e51448; 2012.[divider scroll_text=”SCROLL_TEXT”]

Identification of host genes and pathways that influence resistance or susceptibility to Toxoplasma.

Host genetic differences also determine the outcome of infection with Toxoplasma. Using rat strains that are resistant or susceptible to Toxoplasma we found that macrophages from resistant rats rapidly die following Toxoplasma infection, which prevents parasite replication. This rapid macrophage cell death is mediated by a cytosolic innate immune receptor, NLRP1, which activates caspase-1/11 (inflammasome activation). In mouse macrophages, Toxoplasma also activates the inflammasome but this does not lead to macrophage death and therefore Toxoplasma can replicate freely. Because NLRP1 plays a role in human differences in susceptibility to Toxoplasma we are currently determining the exact mechanism by which Toxoplasma activates NLRP1.
Different mouse strains have different susceptibilities to Toxoplasma infection. We determined the transcriptomes and functional responses of macrophages from recombinant inbred mouse strains that differ in their response to infection or other immune stimuli. We then mapped the loci that determine strain differences in: 1) Toxoplasma killing; 2) the inflammatory response; 3) RNA splicing; and 4) RNA editing rates. We found that differential splicing of the RNA-editing enzyme Apobec1 determines mouse differences in RNA editing activity. We are using genetic approaches to validate other mouse candidate genes involved in these macrophage differences. With collaborators we are exploiting this transcriptome dataset to identify the mouse genes that determine individual differences in resistance to Trypanosoma cruzi and Francisella tularensis. Ultimately, the best strategy for Toxoplasma is to keep the host alive and not cause too much harm as only the chronic parasite stage can be transmitted to new hosts. In other words, if Toxoplasma kills its host, it dies with it. However, the Toxoplasma proteins that modulate the host cannot work optimally in all the different host species it can infect. Therefore, a Toxoplasma strain that has adapted to a specific host species might cause disease and death in a different host species. By investigating which Toxoplasma strain thrives in what host species or strain we hope to identify the Toxoplasma and host proteins that determine their co-evolution.
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  • Hassan MA, Saeij JP. Incorporating alternative splicing and mRNA editing into the genetic analysis of complex traits. Bioessays. 36(11):1032-40; 2014.
  • Gorfu G$, Cirelli KM$*, Melo MB, Mayer-Barber K, Crown D, Koller BH, Masters S, Sher A, Leppla SH, Moayeri M#, Saeij JP#, Grigg ME#. A dual role for inflammasome sensors NLRP1 and NLRP3 in murine resistance to Toxoplasma gondii. mBio. 5(1) e01117-13; 2014.
  • Cirelli KM*, Gorfu G, Hassan MA, Printz M, Crown D, Leppla SH, Grigg ME#, Saeij JP#, Moayeri M#. Inflammasome sensor NLRP1 controls rat macrophage susceptibility to Toxoplasma gondii. PLoS Pathogens. 13;10(3):e1003927; 2014.
  • Hassan MA, Butty V, Jensen KD, Saeij JP. The genetic basis for individual differences in mRNA splicing and Apobec1 editing activity in murine macrophages. Genome Research. 24(3):377-89; 2014.[divider scroll_text=”SCROLL_TEXT”]

The role of sexual recombination in Toxoplasma population structure and the emergence of virulence

Toxoplasma can propagate through entirely asexual means in its hosts. However, when two different Toxoplasma strains are present in an animal that is eaten by a feline, sexual recombination can generate a huge number of genetically distinct organisms that will contain different combinations of the proteins that modulate the host cell compared to the original parental strains. Some of these new strains may have enhanced fitness in different animals than the original parental strains and therefore could cause chronic infections in a wide-range of new animals while others might cause more severe disease. However, it is not easy for two strains to infect the same animal since infection with the first strain is believed to cause immunity against the second strain. This likely explains why just a small number of largely clonal Toxoplasma strains exist in North America and Europe and their combination of proteins are likely adapted to the domesticated cat-house mouse niche. Which factors determine parasite population structure, how certain parasite clones come to dominate a population and which factors drive the success of certain clones are major unsolved questions.
We discovered that South American strains excel at infecting an animal already infected with another strain (superinfection). This leads to sexual recombination when that animal is eaten by a feline and likely explains the high diversity of strains present in South America. We determined that the evasion of IFNγ-mediated killing by the ROP5 and ROP18 virulence factors is necessary, but not sufficient, for superinfection. We are currently using crosses between a strain that can superinfect and a strain that cannot to identify additional Toxoplasma genes that determine superinfection ability. By identifying SNPs in many strains and using these to create a Toxoplasma haplotype map we found that sexual recombination plays a major role in the evolution of Toxoplasma strains and the shaping of Toxoplasma population structure. Many strains share haploblocks and these strains appear to have formed through recent recombination events. We found that the most common North American clonal lineage, although rarely isolated in South America, has introgressed with many South American strains. Three haploblocks had recent common ancestry across multiple diverse lineages, suggestive of a selective sweep driven by fitness loci in those regions. The ROP5 effector gene cluster was in one of these haploblocks indicating that specific ROP5 alleles might confer enhanced fitness in the wild. We are now using this haplotype map to identify associations between haploblocks and specific phenotypes such as disease severity or fitness in a specific host niche. Thus, differences in superinfection-ability leading to frequent (South America) or rare (North America) sexual recombination among Toxoplasma strains may be important in determining global Toxoplasma population structure.
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  • Jensen KD$, Camejo A$, Melo MB, Cordeiro C, Julien L, Grotenbreg GM, Frickel EM, Ploegh HL, Young L, Saeij JP. Toxoplasma gondii Superinfection and Virulence during Secondary Infection Correlate with the Exact ROP5/ROP18 Allelic Combination. MBio. 6(2). pii: e02280-14. 2015.
  • Minot S$, Melo MB$, Li F, Lu D*, Niedelman W*, Levine SS, Saeij JP. Admixture and recombination among Toxoplasma gondii lineages explains global genome diversity. PNAS. 14;109(33):13458-63; 2012.
  • Boyle, JP, Rajasekar B, Saeij JP, Ajioka JW, Berriman M, Paulsen I, Roos DS, Sibley D, White M, Boothroyd JC. Just one cross appears capable of dramatically altering the population biology of a eukaryotic pathogen like Toxoplasma gondii. PNAS 103(27): 10514-10519; 2006.
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