Toxoplasma gondii, the causative agent of toxoplasmosis, is the most prevalent persistent human parasitic infection. The obligate intracellular parasite can invade any nucleated cell residing as a tachyzoite in a parasitophorous vacuole (PV). Gamma interferon (IFNγ) produced mainly by CD8 T cells is the major cytokine controlling Toxoplasma by upregulating host effector defences. We currently study the IFNγ-dependent effector responses against Toxoplasma driven by ubiquitin and guanylate binding proteins (GBPs) and characterise CD8 T cell responses during chronic Toxoplasma gondii infection.
I) Ubiquitin-mediated control of Toxoplasma gondii
During bacterial clearance an initial cellular recognition event marks bacteria or the bacterial inclusion with ubiquitin. This ubiquitination event can either lead to the degradation of the cargo or to signaling events inside the host cell. Whether ubiquitin-driven host defence played a role in Toxoplasma control had been unclear.
We find that in mouse embyonic fibroblasts the PV of type II and not type I Toxoplasma is ubiquitinated in an IFNγ-dependent fashion. Most of the ubiquitin is of the K63 and K48 linkage.
IFNγ stimulation of Human umbilical vain endothelial cells (HUVEC) leads to ubiquitin dependent acidification of Toxoplasma type II parasitophorous vacuoles.
The ubiquitin linkage found around the PV is predominantely of the K63 type and the recruited ubiquitin-binding protein p62 is able to contribute to the restriction of the parasite.
Future questions for the ubiquitin-mediated control of Toxoplasma in human cells:
How is the acidic PV (APV) generated? What Toxoplasma factors mediate the evasion of type I parasites from this pathway? What E3 ligases deposit the ubiquitin? What are the roles of p62 and NDP52 in Toxoplasma restriction?
II) Guanylate Binding Proteins in host defence to intracellular pathogens
Among the proteins highly upregulated as a consequence of interferon-stimulated transcription are the p65 GTPases (GBPs), members of the family of large GTPases that mediate resistance to intracellular pathogens. Contrary to the p47 class of GTPases, the GBPs are present in multiple copies in both the mouse and human genome.
The structure of human GBP1 has been solved and the ability of some family members to hydrolyse GTP to GDP and then to GMP is established. The antimicrobial profile of the human p65 GBPs was restricted to viruses and bacteria and few interaction partners are known.
Human GBP1 in A549 epithelial cells restricts the replication of type II and not type I Toxoplasma and has no impact on Chlamydia and Salmonella.
How does GBP1 restrict type II Toxoplasma in human cells? What Toxoplasma factors mediate the evasion of type I parasites from GBP1-driven control?
III) Properties and function of Toxoplasma-specific CD8 T cells
Through their production of IFNγ, CD8 T cells are important in conferring resistance against Toxoplasma. After an infection with Toxoplasma, both CD4 and CD8 T cells infiltrate the brain. Usually transgenic parasites expressing model antigens in combination with transgenic T cells have been used as models for infection. Obvious shortcomings of this approach include difficulties in controlling expression levels and localisation of the antigen, and moreover the model transgenic T cell receptors might not recognise the model antigen with physiological affinity.
We have identified three CD8 T cell epitopes derived from three distinct Toxoplasma proteins, dense granule protein GRA4, rhoptry protein ROP7 and the unnamed protein T57. Another CD8 epitope from GRA6 has been described. Using flow cytometry to sort antigen-specific CD8 T cells from Toxoplasma-infected mice, transnuclear (TN) Toxoplasma-specific CD8 T cell receptor (TCR) mice for these four Toxoplasma epitope-MHC complexes have been generated by somatic cell nuclear transfer. We have shown that transnuclear CD8 T cells specific to T57 and Gra6 are able to lower the parasite load during the acute phase of Toxoplasma infection.
Interestingly, we have three lines of mice with unique TCR sequences that ROP7 epitope-bound MHC class I albeit with different binding affinity. This enables further studies on how the binding affinity of the TCR to MHC controls CD8 T cell phenotypes and function. We are characterising and comparing natural Toxoplasma antigen-specific CD8 T cells in the chronic phase of Toxoplasma infection in the brain depending on the interaction of different TCRs with the same epitope-major histocompatibility complex.
In the future we are discontinuing this line of research.