Research

OVERVIEW: Our research

Toxoplasma gondii is a protozoan parasite that infects and persists in many warm-blooded animals, from birds to humans.  In some mammalian hosts, including humans and mice, Toxoplasma commonly persists in the central nervous system (CNS) or brain.  Toxoplasma’s ability to cause a long term infection in the CNS requires that the CNS and Toxoplasma avoid an overly-exuberant CNS immune response that could destroy both the host (the CNS) and the microbe.  Our goal is to define the cellular and molecular CNS-Toxoplasma interactions that enable long term CNS persistence. By understanding the molecular mechanisms that underlie persistence, we hope to:  1. identify new targets for treating chronic CNS toxoplasmosis, for which there is currently no therapy, and 2. define new pathways for modulating the CNS immune response. The latter goal may help us develop treatments for neurologic diseases and disorders, ranging from Alzheimer’s disease to stroke, in which CNS inflammation plays a significant role.

BACKGROUND

THE MICROBE: Toxoplasma gondii

Toxoplasma gondii is a eukaryotic, single cell obligate intracellular parasite that infects up to 1/3 of the world’s human population. Congenital infections with Toxoplasma or reactivation of the parasite in severely immunocompromised patients (e.g. AIDS patients) can lead to devastating neurologic consequences but most people infected with Toxoplasma experience no effects from this long term CNS infection.  Thus, Toxoplasma has evolved mechanisms to avoid provoking a CNS immune response that would lead to its clearance and it is these mechanisms we would like to understand.  In addition to being a medically important neurotropic microbe, Toxoplasma has certain characteristics that make it a great tool for understanding CNS-microbe interactions.  These include:

• Genetic tractability & asexual haploid life cycle i.e. you can knock in and out genes relatively easily. 
• Strain differences in acute and chronic stages these differences will make it much easier to detect how Toxoplasma differentially manipulates host cells and then enables us to determine which Toxoplasma proteins are critical to these manipulations.
• Natural pathogen in mice which allows us to use mice to answer questions about the Toxoplasma-CNS interaction.
• Toxoplasma community a friendly, fun-loving group that likes to collaborate and do science! Out of this group has grown ToxoDB (which is part of Eukaryotic DB) which is database/repository for the ‘omics that are being performed in Toxoplasma.

CNS

The CNS/brain are made up of multiple cell types (e.g., neurons, astrocytes), and like several other CNS-tropic microbes, Toxoplasma primarily persists in neurons. While limited data had previously suggested that this neuronal persistence was because neurons could not clear intracellular parasites, the Koshy Lab’s development of a system that allows tracking of parasite-host cell interactions at the single cell level in vivo (Koshy 2010, Koshy 2012) suggested that Toxoplasma persists in neurons because the parasite primarily interacts with neurons during a natural infection (Cabral, Tuladhar 2016). This system also showed that most neurons that interact with Toxoplasma do not become persistently infected.  These findings marked a major shift in how we think about CNS toxoplasmosis. They suggested that the neuron-Toxoplasma interface is crucial for setting the balance between parasite persistence and an overwhelming immune response that clears the parasite but damages the host. For this reason, the Koshy Lab primarily focuses on understanding neuronal immune response against Toxoplasma and how Toxoplasma counters these responses. One mechanism that Toxoplasma uses to counter or evade host immune responses is to switch from its fast-growing stage (the tachyzoite) to its slow growing stage (the bradyzoite). Bradyzoites persist in the host cell inside a cyst. Like other labs, we have found that neurons in vitro naturally induce tachyzoites to switch to bradyzoites, which then encyst.

Models of CNS toxoplasmosis

To understand CNS-Toxoplasma interactions, we primarily use a couple of models.

Mice: Like humans, mice are natural hosts for Toxoplasma, require the cytokine IFN-γ for Toxoplasma control, and have persistent parasites in the CNS. By using mice, we can understand how complex dynamics between the parasite, neurons, and non-neuronal cells (especially immune cells) influence persistence and the CNS immune response. We often start with observations in mice because that helps us focus on what happens during a natural infection.

Neurons: Once we have an idea about what is happening during a natural infection, we may move into an in vitro neuronal system to work out the molecular mechanisms, which are much easier to do in vitro than in vivo. We use both mouse/murine and human neurons because they have different advantages (many genetically modified mice are available while human neurons allow us to translate our findings in mice to human disease).

CURRENT PROJECTS

1. Defining neuron anti-Toxoplasma defenses in vitro and in vivo

Though we have shown that murine and human neurons mount anti-parasitic defenses, many questions remain unanswered. For example, neuronal and non-neuronal murine cells use similar IFN-γ dependent terminal effector proteins to kill intracellular parasites, but human non-neuronal cells use cell-type specific effectors. Thus, we are using human stem cell derived neurons to molecularly define the IFN-γ dependent mechanisms that human neurons deploy against Toxoplasma. In addition, our in vivo work suggests that neurons may also use IFN-γ independent mechanisms to control Toxoplasma. To address this possibility, we are working on defining the full complement of pathways activated in neurons during a Toxoplasma infection in vivo.

2. Identifying how TGME49_207210, which we call the little one (or TLO), affects stage conversion and long-term persistence.

Understanding how Toxoplasma moves from the tachyzoite form to the bradyzoite form is important for understanding Toxoplasma biology AND ultimately developing drugs that target the bradyzoite. Through bioinformatic analyses of parasites during murine neuron infection we identified TGME49_207210/TLO as highly upregulated in bradyzoites. TLO is a small, hypothetical protein (110 aa) with the only homologs being in other tissue cyst forming Apicomplexa. So far we know TLO matters for stage conversion in vitro and persistence in vivo but we don’t know how TLO functions, which is the focus of our current work.