Our research program is driven by the application of molecular genetic approaches to the understanding of cell signaling in intracellular pathogens with a focus on the parasite Toxoplasma gondii. Toxoplasma is an obligate intracellular eukaryotic parasite with the remarkable ability to infect virtually any nucleated cell from a wide range of mammalian and avian species. Toxoplasma infects approximately a third of the human population and, due to its ability to form a latent stage in various organs, including the brain, the infection persists for the lifespan of the host. New infections or reactivation of latent parasites in the immunocompromised or immunosuppressed can cause serious disease and death. In addition, primary infection during pregnancy can lead to miscarriage and stillbirth. Although there are available medications for the treatment of acute toxoplasmosis, these are poorly tolerated and do not affect the latent form . Thus, this ubiquitous parasite is a significant threat to human health, and the discovery of new therapeutics is a high priority.

Key to discovering novel and effective targets for drug development is the expansion of our understanding of processes that are specific and essential to the parasite. It is within this context that my research team focuses on elucidating the signaling and cellular processes involved in the propagation of the parasite through infected tissue. Overall, our research program has been driven by the following general questions: what are the cues and proteins regulating how the parasite exits its host cell, and how is this parasite able to adapt to the variety of physiological environments and stresses it encounters during its life cycle? Current projects, which are delineated below, include the characterization of the kinases and phosphatases that regulate the propagation cycle of Toxoplasma and the mechanistic study of the morphological changes undergone by the parasite mitochondrion during propagation. Our research team addresses these projects with a combination of molecular genetics, cell biology, and protein biochemistry.

A unique feature of Toxoplasma is the presence of a single tubular mitochondrion, which is essential for parasite survival and a validated drug target. Toxoplasma’s singular mitochondrion is very dynamic and undergoes morphological changes throughout the parasite’s life cycle. Extensive morphological changes also occur as the parasite transitions from the intracellular to the extracellular environment. While inside a host cell, the mitochondrion is maintained in a lasso shape that stretches around the parasite periphery, where it has regions of coupling with the parasite pellicle, suggesting the presence of membrane contact sites. Promptly after exit from the host cell, these contact sites disappear, and the mitochondrion retracts and collapses towards the apical end of the parasite. Prior to our work, neither the functional significance nor the proteins involved were known for the contact between Toxoplasma’s mitochondrion and pellicle. We have identified a novel protein, LMF1 for Lasso Maintenance Factor 1, that is responsible for tethering the mitochondrion to the pellicle of the parasite. Ongoing projects aim to: 1. Determine the biological relevance of LMF1dependent mitochondrial morphology; 2. Identify and characterize components of the LMF1 attachment complex; and 3. Determine functional changes in LMF1 during the lytic cycle.

Our work on Toxoplasma’s mitochondrion has expanded to the investigation of the machinery that maintains the unusual mitochondrial genome. Toxoplasma has a reduced mitochondrial genome consisting of 21 non-random concatenated sequence blocks. The mechanisms by which this unusual structure is maintained and regulated are not known. Previously, we identified a homolog of the mismatch repair enzyme MutS (TgMSH1) that localizes exclusively to the mitochondrion. Deletion of this gene results in the accumulation of single-base mutations in mtDNA and a reduction in mtDNA content. We have applied nanopore sequencing to the mtDNA and confirmed our observations that disruption of TgMSH1 leads to a reduction in mtDNA content and accumulation of transition mutations. Intriguingly, in the absence of TgMSH1, we observe an accumulation of consecutive repeats of specific sequence blocks, which results in longer mtDNA molecules. Thus, TgMSH1 plays a critical role in the maintenance of the unique architecture of the mtDNA, and its study might reveal the mechanisms behind the unusual makeup of the mitochondrial DNA. Future work includes identifying functional partners of TgMSH1, determining the role of the various functional domains of TgMSH1, and using TgMSH1 as a tool to determine the structure of the mtDNA.

The propagation cycle of Toxoplasma is exquisitely regulated by reversible phosphorylation. As illustrated by our previous work with TgCDPK3, much is known about the unique kinases and their substrates in Toxoplasma. By contrast, little is known about the role of phosphatases. To begin addressing this knowledge gap, we inventoried protein phosphatases in Toxoplasma gondii and related parasites. Based on this work we characterized serine/threonine protein phosphatases predicted to be membrane-associated and determined that only PPM5C, a PP2C family protein phosphatase, localizes to the plasma membrane, where it regulates attachment to host cells. We have also characterized the Toxoplasma homolog of Phosphatase of Regenerating Liver (PRL), which associates with the parasite plasma membranes via a conserved prenylation site. Disruption of TgPRL results in a defect in the parasite’s ability to attach to host cells and, most importantly, complete loss of virulence in mice. Immunoprecipitation experiments revealed that the PRL-CNNM (cyclin M) complex, which regulates intracellular magnesium homeostasis in mammalian cells, is also present in Toxoplasma. Consistent with this interaction, we showed that TgPRL is involved in regulating intracellular magnesium homeostasis. We are currently characterizing the role of PRL in the chronic stage of the parasite, as well as its regulation during the lytic cycle of the parasite. We have also characterized the Kelch-like domain-containing phosphatase PPKL, which is unique to the parasite and absent in its mammalian host. We determined that PPKL is essential and plays a critical role in parasite daughter formation. Phosphoproteome analysis, in combination with the identification of PPKL interactors, has revealed a network of signaling proteins that regulate parasite division and daughter cell formation.