Research in the Saeij lab

Parasites and their hosts co-evolve, leading to variation in both host and parasite genomes and to the emergence of differences in parasite virulence and host resistance.  We are interested in host-parasite interactions between the obligate intracellular eukaryotic parasite Toxoplasma gondii and its mammalian hosts.  One of our long-term goals is to understand how distinct Toxoplasma strains differ in their ability to cause disease in humans. The focus of my laboratory over the last years has been to identify genes of Toxoplasma that determine virulence, host genes and pathways that determine resistance/susceptibility, and to characterize their specific interactions. To achieve this we use a combination of genomics, biochemistry, genetics, microscopy, immunology and computational tools.

Toxoplasma proteins that modulate the immune response

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. 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.

 

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.

 

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. Other groups have shown that this is mediated by the Toxoplasma secreted effector TgIST. We recently performed Toxoplasma whole genome CRISPR/Cas9 loss-of-function screens to identify Toxoplasma genes that determine fitness in interferon-gamma stimulated human cells.

 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. 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.