The major goal of this project is to determine the impact of gene duplication and expansion on the ability of the human opportunistic pathogen, Toxoplasma gondii, to cause severe disease in HIV/AIDS patients. HIV/AIDS patients are at risk from severe disease caused by Toxoplasma due to 1) primary infection with environmentally stable infectious oocysts, 2) primary infection from tissue cysts present in undercooked meat and 3) reactivation of dormant tissue cysts due to a lack of immune surveillance due to HIV/AIDS-driven immunosuppression. These tissue cysts, and particularly the effector proteins that they secrete during interactions with the host, are the primary focus of this proposal. We find this work particularly importance since in contrast to the acute phase of infection, much less is known about the T. gondii effectors required for T. gondii to cause disease in the chronic phase of infection. This work builds off of our previous studies identifying tandemly duplicated and diversified genes that play a key role in the acute virulence of T. gondii in an animal model of infection. Extending upon this result, we have identified and thoroughly curated all tandemly expanded gene clusters in the T. gondii genome. Two important pieces of data emerged from this study: T. gondii expanded gene clusters are significantly enriched 1) for genes predicted to be secreted from the parasite (and therefore interact with the host cell) and 2) genes that are expressed during the latent cyst stage that is so crucial for causing disease in HIV/AIDS patients.
In Aim 1 we fully characterize these Cyst-Expressed Expanded Loci (CEELs) in terms of their total gene content across multiple parasite strains and clone and sequence the genes that they encode. We use epitope tagging to determine their putative location within the parasite and host cell. Those loci that are secreted from the parasite will then be the focus of Aim 2.
This Aim i s facilitated by our extensive experience characterizing expanded loci in T. gondii and will provide important new information about their gene content, information which will be useful to us and to the broader Toxoplasma research community.
In Aim 2 we will perform gene knockouts for loci selected from Aim 1 and determine their impact on interaction with the host, with a strong focus on cyst biology. We will determine the effect of CEEL deletion on cyst infectivity, formation, and the ability of latent cyss to reactivate in a well-established mouse model of infection. Successful completion of these experiments will identify new Toxoplasma cyst-expressed effectors that play a key role in disease in HIV/AIDS patients. In future studies we will characterize their mechanism of action.
Toxoplasma is an important opportunistic pathogen in HIV/AIDS patients. Over 1 billion humans have been infected worldwide, putting an enormous number of untreated or poorly treated HIV/AIDS patients at risk for this disease. Our goal is to use an integrative approach to identify new Toxoplasma genes that are responsible for causing disease in these patients.
|Lorenzi, Hernan; Khan, Asis; Behnke, Michael S et al. (2016) Local admixture of amplified and diversified secreted pathogenesis determinants shapes mosaic Toxoplasma gondii genomes. Nat Commun 7:10147|
|Wier, Gregory M; McGreevy, Erica M; Brown, Mark J et al. (2015) New method for the orthogonal labeling and purification of Toxoplasma gondii proteins while inside the host cell. MBio 6:e01628|