Toxoplasma gondii, Cryptosporidium spp and malaria parasites are unified by their phylogenic relationships and by their life threatening impact on individuals with HIV/AIDS. Malaria resistance to chloroquine is conferred by mutations in chloroquine resistance transporter (PfCRT), an integral membrane protein capable of shuttling chloroquine out of the malaria digestive vacuole (DV) where it inhibits heme detoxification. We recently discovered that T. gondii expresses an ortholog of PfCRT termed TgCRT, and that this putative transporter is associated with a T. gondii digestive organelle termed the vacuolar compartment or VAC. Targeted deletion of TgCRT causes bloating of the VAC, affects parasite replication, attenuates parasite virulence and results in a 90% decrease in neural tissue cysts during chronic infection. In malaria, CRT has been implicated it in the transport of glutathione and the regulation of redox potential, but these studies were done with chloroquine resistant mutants of PfCRT. The inability to ablate PfCRT has also impeded efforts to understand the native function of this important protein. Virtually nothing is known about the redox regulation in T. gondii despite its role in combatting oxidative stress during infection. Herein we propose to exploit the genetic tractability of T. gondii to dissect the function of TgCRT by implementing for the first time in T. gondii redox biosensors capable of measuring redox status in relevant compartments and testing the impact of TgCRT ablation on the regulation of redox potential and susceptibility to oxidative stress. We will also identify proteins that associate with TgCRT and/or are proximal to it as a means of defining its relationships with other proteins potentially involve in redox regulation. Demonstrating that TgCRT confers resistance to oxidative stress will create new avenues to assessing the importance of oxidative stress in controlling T. gondii infection. Showing that the T. gondii VAC contributes to redox regulation will strengthen the evolutionary link between this organelle and the malaria DV. Together the findings have implications for future antagonism of the VAC and redox pathways to affect the outcome of HIV/AIDS associated diseases including toxoplasmosis, cryptosporidiosis and malaria.
The protozoan parasite Toxoplasma gondii causes severe disease in HIV/AIDS individuals with weakened immunity. Current therapies have significant limitations, necessitating a better understanding of how this parasite survives, and thrives, during infection. The proposed studies are expected to yield insight into how T. gondii is resistant to a malaria drug called chloroquine and how protects itself against oxidative damage by regulating an anti-oxidant pathway. The findings have the potential to open new opportunities to interfere with the parasite anti-oxidant pathway improve the clinical management of infection.
| Lunghi, Matteo; Spano, Furio; Magini, Alessandro et al. (2016) Alternative splicing mechanisms orchestrating post-transcriptional gene expression: intron retention and the intron-rich genome of apicomplexan parasites. Curr Genet 62:31-8 |
