Discovering new factors controlling virulence in Toxoplasma gondii – ToxoVirFT

Our goal is to identify and characterize the crucial regulators of the tachyzoite virulence by discovering the transcription factors (TF) that control the expression of the rhoptries and micronemes transcripts and proteins. Our strategy is devised to mix ambitious steps identifying unknown DNA elements regulating the timing and strength of key virulence factor's promoters and the purification of TF bound to those specific DNA. We will also perform a more straight forward characterization of proteins with clear TF potential selected based on their ability to interact with a known regulator of those virulence factors. Those two complementary strategies will allow us to find new proteins of previously unknown function with an unbiased approach and also clarify the involvement of known potential TF in the control of virulence. The identification and the characterization of those regulators will rely of the use of cutting edge technology including proteomics, transcriptomics and reverse genetic. We aim at discovering the regulators, their interaction web with other proteins and their regulatory network.

We identified transcription factors that are able to interact with another known transcription factor described as a regulator of virulence gene because of its ability to bind to promoters of these genes. The expression profile of these new factors correspond to exact timing of expression of the virulence genes. The direct targeting of one of these factors showed that it may be responsible of the expression of virulence genes. Moreover, the mutant parasites show an impairment in invasion of host cell, a key process for virulence in vivo.
We are also in the process of identifying DNA motifs critical for the expression of virulence genes.

Gene regulation is poorly understood in Apicomplexa. Only few transcriptional regulators and cis acting elements have been characterized so far.
Our goal is to identify and characterize the crucial regulators of the tachyzoite virulence by discovering the transcription factors (TF) that control the expression of the rhoptries and micronemes transcripts and proteins.
Detailed characterization of their interaction web and regulatory network will provide new insights into the pathways regulating virulence as well as cell cycle. Indeed, virulence factors expression is strictly controlled during the cell cycle in concordance with the creation of organelles they are packaged into. As a final result of this study, we wish to gain more knowledge on cell cycle regulation by linking the discovered TF to the regulators that orchestrate the cell cycle.
Discovering those regulators will also represent an important step into the understanding of the molecular mechanism involved in the timing of the establishment of organelles during the cell cycle. It may also shed a new light on related steps in other Apicomplexa, as Plasmodium.
Moreover, because the regulators identified are likely to be Apicomplexa-specific the accumulated knowledge during the course of this study may be a first step toward the discovery of new drug targets and therapeutic strategies.

Manuscripts will be written and submitted in the the future.

The life cycle of T. gondii is complex with multiple proliferation and differentiation steps that are critical to survival of the parasite in its human and feline hosts. The tachyzoite proliferation is the most relevant step for pathogenesis in humans. Tachyzoites express invasion and virulence factors that are crucial for their survival in the host. The expression of those factors is tightly controlled during the tachyzoite cell-cycle to permit their correct packaging in the newly formed organelles (micronemes and rhoptries) in the daughter cells. However, little is known about the actors that control the gene expression of those important factors. Only few transcriptional regulators and DNA motifs have been characterized so far in T. gondii. Our goal is to identify and characterize the crucial regulators controlling gene expression of the invasion and virulence factors. We will use de novo identification of DNA motifs controlling the timing of expression of those genes. We will purify the proteins specifically bound to those motifs. Proteomics will then allow the identification of the proteins potentially involved in the gene regulation of those important virulence factors. Using reverse genetic, transcriptomics and proteomics, we plan to characterize those factors and reveal their connections with the signals controlling the cell-cycle progression.