Artificial Regulation of Transcription in Toxoplasma gondii, The Model Apicomplexan
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(Thesis)
Date
2021
Authors
Sharifpour, Mohammad Farouq
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Advisors
McAllister, Milton M
Hemmatzadeh, Farhid
Hemmatzadeh, Farhid
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Abstract
Toxoplasma gondii is an intracellular protozoan parasite that can infect all warm-blooded animal species. Toxoplasma has substantial impacts on human and animal health. Acute toxoplasmosis causes various problems such as neural and congenital disorders. This is however, only the tip of the iceberg as most of the infections persist in a latent form that is now recognised to be a major risk factor for numerous conditions such as schizophrenia. Beside the importance of acute and latent infections by itself, Toxoplasma is also a valuable research model for the biology of other protozoa within the phylum Apicomplexa, e.g. Plasmodium, Cryptosporidium, Babesia, Theileria and Eimeria. Transcription is a critical step in the regulation of gene expression. Artificial transcription factors have been developed to artificially regulate transgenes or native genes. This artificial regulation can be beneficial in many ways, including molecular biology, biotechnology and gene therapy. In my PhD project, I pursued two major aims. First, I enhanced the regulatory feature of the existing Tet-responsive transcription system of Toxoplasma, aka TATi. TATi is an allosteric regulatory system for transcription in Toxoplasma that has been in use for about 20 years. Despite having a record of success in T. gondii research, TATi induces relatively high background noise. I decided to attempt to modify the system in Toxoplasma, with a primary goal of reducing background noise. I managed to dramatically increase the signal to noise ratio (23-fold) of the TATi system by employing a new minimal promoter element (GRA2-MP) as one of the system's building blocks. GRA2-MP improved the TATi system's efficiency by reducing background noise when the system was switched ‘off’ and by increasing the system's transactivating capability when the system was switched ‘on’. The superiority of the new minimal promoter in the context of the TATi system was convincingly demonstrated in all replicates of both transient and stable transfection experiments. The second major aim of my PhD project was to build a foundation for a new transcription regulatory system by developing a novel modular artificial transcription factor (ATF) in Toxoplasma gondii as a model organism of apicomplexan parasites including Plasmodium and others. Pursuing this goal, I designed a series of complex ATFs with modular features including DNA binding (DBD) and transactivating (TA) domains. This new transcription regulatory system incorporates nine different elements and modules. As demonstrated using transient and stable transfection experiments, a functional prototype for a novel ATF was successfully established in Toxoplasma, consisting of a DBD from Saccharomyces cerevisiae and a Plasmodium falciparum-derived TA. Creation of a new ATF was a goal with an inherent high risk of failure because, like a chain composed of links, each and every one of the numerous elements and modules must function properly for the overall ATF to be able to initiate transcription. Any ATF candidate that fails to initiate transcription can be difficult to troubleshoot, because the problem might be associated with any link in the chain or could even involve multiple links. By developing a functioning ATF, which I have named GP, a huge hurdle of creating a functional ATF was overcome. Thanks to its modular feature, GP is fully amenable to future improvements by testing the replacing of any module one at a time. GP is also equipped with FKBP and FRB elements that can be used in the future to endow dimerization within a rapamycin regulated transcription system (RRTS). Availability of an RRTS will provide desirable advantages over TATi for future investigations of apicomplexan parasites, e.g. having drug-on rather than a drug-off transcription control and possessing a Nuclear Localisation Signal to further ensure tighter regulation of transcription.
School/Discipline
School of Animal and Veterinary Sciences
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Animal and Veterinary Sciences, 2021
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