Eva Frickel

 

Immune-mediated killing of toxoplasma gondii in stem cell derived macrophages

The parasite Toxoplasma gondii infects host cells by an active invasion process and resides within a parasitophorous vacuole (PV. Infection by Toxoplasma leads to production of the proinflammatory cytokine interferon gamma (IFN), which stimulates cell-autonomous defence mechanisms to kill and restrict the pathogen within the PV. We have identified two effector pathways of IFN-dependent killing of Toxoplasma in human endothelial and epithelial cells. Since macrophages are one of the most important innate immune defence cell types, we have set up human stem cell derived macrophages and their genetic manipulation by siRNA. This project will investigate the mechanism of anti-Toxoplasma control in stem cell macrophages and compare them to the pathways we have previously identified in endothelial and epithelial cells.

In IFN-stimulated human endothelial cells, the PV of type II, but not type I Toxoplasma is decorated with the cellular protein ubiquitin (1). Ubiquitination of intracellular pathogens can lead to autophagy-dependent clearance of the pathogen via host adaptor proteins (2). We indeed find p62 and NDP52, two such autophagy adaptor proteins, at the PV of avirulent Toxoplasma. In spite of this, in endothelial cells, destruction of Toxoplasma is mediated by non-autophagic acidification of the PV with characteristics of the endo-lysosomal pathway. 

IFN upregulates the p65 Guanylate Binding Proteins (GBPs). The GBPs are a family of GTPases that are conserved amongst vertebrates and 7 Gbps have been identified in humans. Mouse GBPs have been shown to restrict Toxoplasma by disrupting the parasitophorous vacuole (PV) (3). However, we have demonstrated that human GBPs are not recruited directly at the PV in epithelial cells yet in GBP1 CRISPR knockout human epithelial cells, IFN-mediated cell autonomous killing of Toxoplasma is impaired (4).

Using human stem cell macrophages, you will investigate if

1) autophagy and/or ubiquitin-driven mechanisms can eliminate Toxoplasma and

2) if GBP-driven alteration of autophagy and/or cellular metabolism plays a role in Toxoplasma destruction. 

For the first part of the project you will work with autophagy knockout and reporter stem cell macrophages that we have obtained and determine the effect on Toxoplasma killing and replication. For this you will use a recently set-up high content imaging platform to conduct automatic measurement of percent infected cells and measure Toxoplasma PV size. If a dependence of Toxoplasma restriction by autophagy is identified, you will determine which compartment the parasite is contained in is targeted (vacuole versus phagosome), you will study the kinetics of the restriction and identify essential players for this pathway by targeted siRNA of candidate genes.     

For the second part of the project, you will investigate if knockdown of various human GBPs has an effect on cellular metabolism and autophagy and thereby on Toxoplasma growth. Specifically, you will test if GBPs have an effect on Indoleamine 2,3-dioxygenase (IDO) - an enzyme that catabalises tryptophan and is essential in Toxoplasma control. Additionally, you will probe if GBPs alter the metabolic state of the cell via mTor.

In summary, this project will lead to the enhanced understanding of IFN-mediated restriction of Toxoplasma in human cells.   

1. Clough, B., Wright, J. D., Pereira, P. M., Hirst, E. M., Johnston, A. C., Henriques, R. and Frickel, E.-M. (2016)
K63-linked ubiquitination targets Toxoplasma gondii for endo-lysosomal destruction in IFNγ-stimulated human cells.
PLOS Pathogens 12: e1006027. PubMed abstract

2. Sorbara, M. T. and Girardin, S. E. (2015)
Emerging themes in bacterial autophagy.
Current Opinion in Microbiology 23: 163-170. PubMed abstract

3. Yamamoto, M., Okuyama, M., Ma, J. S., Kimura, T., Kamiyama, N., Saiga, H., Ohshima, J., Sasai, M., Kayama, H., Okamoto, T., Huang, D. C. S., Soldati-Favre, D., Horie, K., Takeda, J. and Takeda, K. (2012)
A cluster of interferon-γ-inducible p65 GTPases plays a critical role in host defense against Toxoplasma gondii.
Immunity 37: 302-313. PubMed abstract

4. Johnston, A. C., Piro, A., Clough, B., Siew, M., Virreira Winter, S., Coers, J. and Frickel, E.-M. (2016)
Human GBP1 does not localize to pathogen vacuoles but restricts Toxoplasma gondii.
Cellular Microbiology 18: 1056-1064. PubMed abstract