Toxoplasma Research Lab: Investigating Host-Parasite Interactions

Toxoplasma Research Lab: Unraveling Host-Parasite Interactions

Our Toxoplasma Research Lab is dedicated to understanding the complex host-parasite interactions between Toxoplasma gondii, an obligate intracellular eukaryotic parasite, and its mammalian hosts. The focus of our laboratory over the last years has been to identify Toxoplasma genes that determine virulence, host genes and pathways that determine resistance or 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 determine in vivo fitness

To establish a chronic infection in its host, Toxoplasma needs to survive at the site of infection, disseminate, cross biological barriers, and evade immune responses. To do this, Toxoplasma uses secreted effector proteins called ROPs and GRAs, which are secreted into the host cell from Toxoplasma secretory organelles called rhoptries and dense granules. We have established in vivo CRISPR/Cas9 loss-of-function screens that can identify Toxoplasma genes involved in these processes.

Dissemination: Toxoplasma‘s pathology mainly relies on the dissemination of the parasite from the site of infection to various essential organs, such as the brain, where it causes tissue destruction. Toxoplasma often uses a Trojan Horse mechanism to facilitate its dissemination, during which it hijacks the host cell migration machinery to co-opt host leukocytes as shuttling vectors. Exactly how Toxoplasma does so, however, is largely unknown. In a collaboration with Antonio Barragan‘s group, we recently identified a novel Toxoplasma protein important for its dissemination, which we named TgWIP. We found that upon invasion the parasite secreted TgWIP into the host cell cytosol, where TgWIP stimulated dendritic cells to become hyper-migratory and undergo a mesenchymal to amoeboid transition (MAT). The process was associated with a dramatic rearrangement of the actin cytoskeleton. Together with the Stone Chen lab, we are determining the molecular mechanisms by which TgWIP mediates Toxoplasma dissemination in the host.

Evading host immune responses: 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) activates a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identified ~500 Toxoplasma genes that determine parasite fitness specifically in naïve or IFNγ-stimulated murine macrophages. One of these genes encodes dense granule protein (GRA45), which possesses a predicted chaperone-like function and is critical for preventing other GRA effectors from aggregating. Parasites lacking GRA45 mislocalize GRA effectors upon secretion, have enhanced susceptibility to IFNγ-mediated growth inhibition, and have reduced virulence in mice. We are exploring the function of other Toxoplasma genes that determine fitness in IFNγ-stimulated macrophages.

Modulating 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 characterizing novel ROPs and GRAs that came up as hits in our CRISPR/Cas9 screens and are 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 determined that Toxoplasma infection induces cell death of interferon-gamma stimulated human fibroblasts, which is concomitant with early parasite egress and limits parasite replication. We have used CRISPR/Cas9 screens to identify the Toxoplasma genes that determine fitness in interferon-gamma stimulated human fibroblasts and are currently determining the mechanism by which the top hits affect Toxoplasma fitness.

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 IFNγ stimulated human cells. This screen has identified novel GRAs and ROPs and we are currently determining the mechanism by which they function.

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.

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.