Lecture Summaries

1 Introduction

The first session will begin by introducing the instructors to the students and vice versa. We would like to learn why students have chosen to join the course and what they expect to learn and get from it. This session aims to establish the group to help students feel comfortable engaging with one another and with the instructors.

Then, the instructors will give a brief introduction about the course and its different aspects. Finally, the last part of the session will be used to introduce students to the scientific literature and its formats (primary research articles vs. reviews), to approach the use of primary literature using PubMed and MIT library resources.

2 Alien invasion: How Toxoplasma secretly enters host cells

Cells must take up material from their surrounding, both as a way to take up nutrients (endocytosis) and to engulf and destroy pathogens (phagocytosis). Generally, material taken up in either path is targeted for degradation by the lysosome, an acidic organelle containing proteolytic enzymes. The material or pathogen is enclosed in a membrane bound compartment decorated with protein markers. These markers regulate the fusion of the compartment with a sequence of small vesicles, leading to the fusion with the lysosome and destruction.

For intracellular pathogens to survive within the cell, they must first subvert this cellular process. Various strategies have been employed, from escaping the membrane bound compartment (Listeria), subverting host cell trafficking (Legionella) to replicating within the lysosome (Mycobacteria).

The eukaryotic parasite Toxoplasma gondii employs a unique strategy. While most pathogens can only infect one or two types of cell from one species, Toxoplasma is able to invade any cell type from any warm-blooded animal. It is able to do this because instead of relying its host cell to engulf it, Toxoplasma uses its own power to push into a host cell, wrapping itself in the host cell plasma membrane, which becomes the parasitophorous vacuole (PV).

This is the topic of the first paper of the session, using a variety of microscopy techniques, Morisaki et al. (1995) show that T. gondii actively invades both phagocytic and non-phagocytic cells in a parasite-dependent manner. However, invasion is not suffient, the parasite still has to avoid fusion with the lysosome which would destroy it. To do this, during invasion the parasite creates a tight junction between itself and the host cell plasma membrane. Then, Toxoplasma is able to 'sort' host cell proteins, excluding any which would promote the fusion of the PV to the lysosome. Mordue et al. (1999) were the first to observe this unique phenomenon using a selection of fluorescent host cell proteins; they show that some are included while others are excluded from the PV.

3 Make yourself at home: Intracellular survival in the parasitophorous vacuole

Once established within a parasitophorous vacuole (PV) the parasite must actively modify the PV to create a safe environment to replicate and thrive. This modification is of particular importance for Plasmodium, the causative agent of malaria. By infecting red blood cells, which lack internal organelles and no longer actively synthesize membranes, Plasmodium must secrete and target its own proteins to the PV.

From today's first paper by Eksi and Williamson, we will learn that a specific amino acid sequence is needed for proper protein targeting into the PV membrane. The authors fuse the green fluorescent protein (GFP) to different portions of a protein known to be exported to the PV membrane to understand how targeting is regulated. This approach enables them to report the amino acid sequence required for protein targeting to the PV membrane.

The second paper of the session discusses Toxoplasma, which secretes a number of proteins from small vesicles called dense granules into the host cell. Using an elegant approach, Nadipuram et al. (2016) recently discovered a number of novel dense granule proteins localized to the PV of Toxoplasma. By fusing a known PV protein (GRA17) to an enzyme, BirA, all proteins that came into close contact with GRA17 could be isolated and identified. Using this method, the group discovered a number of novel proteins, at least one of which was shown to have an important role in virulence. These papers show how cell biological approaches can be adapted to parasites to determine novel biology.

4 How parasites transport proteins and nutrients across membranes

Any parasite that remains sequestered within a vacuole needs a mechanism by which it can export proteins to the host cell and import essential nutrients. Recently, the protein complexes responsible for this in both Plasmodium and Toxoplasma have been elucidated.

In the first paper of the week, Elsworth et al. (2014) discover this complex in Plasmodium. Named PTEX (Plasmodium translocon of exported proteins), they show that several members of this complex are essential for parasite protein export to the host cell and show that protein export is essential for the parasite's replication. As well as protein export, parasites must be able to take up nutrients from the host cell through the expression of transporters on the parasitophorous vacuole membrane to allow the passage of small molecules.

Gold et al. demonstrate that two proteins secreted by Toxoplasma, GRA17 and GRA23, are important for nutrient access and permeability of the vacuole membrane. This study provided a molecular basis for the existence of pores at the surface of the parasitophorous vacuole membrane and provided evidence for how Toxoplasma is able to survive and proliferate inside an intracellular vacuole.

5 The transforming parasite: How Theileria modifies its host cell in order to replicate

Theileria is another member of the family of parasites that includes Toxoplasma and Plasmodium. This pathogen is transmitted by ticks and causes theileriosis in humans and cattle. In humans, the disease is usually asymptomatic, but can cause high fevers and destruction of a large number of blood cells. In cattle, mortality rates vary depending on the strain of the parasite. Nevertheless, Theileria infection in cattle is a major obstacle to the economic development of affected rural areas. After getting access to the bloodstream following a tick bite, the parasite selectively infects lymphocytes and associates with the host cell microtubules. Lymphocytes infected with Theileria start showing characteristics that are shared with some cancers, including immortalization and a rapid, uncontrolled proliferation. This is a striking and unique example of a eukaryotic cell transforming another eukaryotic cell into a cancer-like state in a process driven by the parasite.

Today we will learn that Theileria can hijack several signaling pathways to establish and maintain host lymphocyte transformation. Dessauge et al. provide the first line of evidence by showing that Theileria is able to increase the transcription and stability of the c-Myc oncogene, inducing the survival of the infected cells. Marsolier et al. identify one of the molecular effectors that the parasite employs to co-opt the oncogenic signaling pathways of it host cell. The authors identify TaPIN1, a prolyl isomerase that promotes transformation and can be specifically blocked with an anti-parasite drug. We will use these to studies to cover the signaling pathways hijacked by Theileria and discuss the bioinformatics approaches used to identify TaPIN1.

6 (Don't) Divide and Conquer: How Toxoplasma pauses the host cell cycle

The host cell cycle poses a challenge for intracellular parasites, the extensive reorganization and cytokinesis that occurs during division could have detrimental effects on the parasite's replication or even survival. As we covered in the previous session, parasites have evolved various strategies to control and manipulate host cell division for their own best interests.

In this session we will examine how Toxoplasma faces this challenge. In the first paper, Brunet et al. (2008) demonstrate that Toxoplasma prevents host cell proliferation by arresting the cells in G2 phase. Interestingly, parasite replication was blocked if the host was allowed to divide. This finding suggests that Toxoplasma requires proliferation arrest to survive, although the reasons for this remain unclear.

The second paper from Bougdour et al. (2013) offers a potential mechanism for this growth arrest. Many proteins are secreted by the parasite into the host cell where they have a number of effects ranging from modulating the immune system (covered in session VII), allowing the uptake of nutrients (session IV) to altering transcriptional responses.

Bougdour et al. (2013) demonstrate that the parasite protein GRA16 is trafficked to the host nucleus, where it interacts with a number of host cell proteins known to be involved in the cell cycle. Modulation of the levels of these host proteins, including the transcription factor p53, appears to prevent the host cell cycle from continuing. This study shows the first details of how the parasite is able to subvert the host cell cycle to its own advantage.

7 Field trip

The tools used to study the biology of parasites have advanced considerably in the past decade, and active research is continuing to improve these methods. We will spend one session visiting the Lourido lab at the Whitehead Institute, where various aspects of the research concerning Toxoplasma gondii discussed in class are currently being performed.

Students will attend hands-on demonstrations of several techniques for visualizing the parasite including fluorescence microscopy and plaque assays. Time permitting, fluorescence-activated cell sorting (FACS) will be discussed and demonstrated.

8 Jamming communications: Strategies to thwart innate immunity

The immune system employs multiple strategies to defend the host from potential invaders. The innate immune system provides the first line of defense, as its components and cells will recognize any potential intruder in a generic manner.

During the first steps of an infection, a particular set of immune cells named dendritic cells will recognize and engulf pathogens before destroying them and migrating to lymph nodes. Once in the nodes, they present the pathogen proteins to other classes of immune cells. This process is called antigen presentation and is a key component in the activation of the second line of defense of the immune system, the adaptive immunity.

During acute infection in humans, Toxoplasma rapidly disseminates to multiple organs. Laboratory studies have shown that after orally infecting mice and rats, the parasite can be found in distant sites hours after invasion. To spread in this way Lambert et al. demonstrate that Toxoplasma seems to employ a "Trojan horse" strategy by hiding inside dendritic cells. Additionally, the parasite also induces a state of hyper-motility in those cells, exploiting the migration of dendritic cells for disseminating through the body of its host.

The paper by Etheridge et al. describes the strategies used by Toxoplasma to overcome the immune system of its host. To defend against intracellular pathogens, cells possess a family of molecules called immunity related GTPases (IRGs), which will bind and break open the intracellular vacuole. Toxoplasma overcomes this important intracellular defense mechanism by secreting and targeting three proteins to its parasitophorous vacuole. These proteins form a complex that will inactivate any IRG oligomer that comes into contact with them, protecting the parasitophorous vacuole from the innate immune response of the host. We will discuss the importance of these strategies used by Toxoplasma to understand pathogen-host interactions and the early steps of innate immune system activation.

9 The fatal consequences of hide and seek: How changes in Plasmodium's surface increases disease severity

One of the key mechanisms that Plasmodium falciparum uses to avoid the immune system is clonal antigenic variation of the family of var genes. By expressing several variants of surface proteins within a population, some parasites can avoid being recognized by host antibodies, allowing them to replicate. This also allows the same host to be infected multiple times, despite producing a good antibody response.

The var gene family encodes PfEMP1, a protein transported to the surface of the infected red blood cell (RBC), which mediates the binding of the RBC to blood vessels. This interaction prevents the infected cells from being cleared by the spleen and is important in malaria pathogenesis. Lavsten et al. (2012) use clinical samples to demonstrate that a specific variant of PfEMP1 is associated with severe malaria symptoms in children.

This finding is further examined by Avril et al. (2012), in the same issue of the journal, who show, using a variety of biochemical and cell biology methods, that parasites expressing this PfEMP1 variant have greater affinity for blood vessels found in the brain as well as having a generally better ability to sequester throughout the body.

10 Malarial mind control–Plasmodium increases transmission by modifying mosquito behavior

To complete its life cycle, Plasmodium needs to cycle between mosquitos and intermediate hosts, such as mammals. It has been suggested that Plasmodium manipulates both mosquitos and its intermediate hosts to ensure its transmission. We will look at two papers investigating different aspects of how the parasite promotes transmission across species.

First, the study by Cator et al. (2013) describes how Plasmodium infection affects the feeding behavior of infected mosquitoes. They show infected mosquitos are more attracted to humans when the parasite is ready to be transmitted (sporozoites) rather than when it is developing. At the same time they show that infection alters the mosquito's sensitivity to human odors.

In the second paper De Moraes et al. (2014), they use a mouse parasite Plasmodium chabaudii to show that mosquitos are preferentially attracted to infected mice. Interestingly, infection alters the odor of the mice and increases the presence of a number of volatile compounds produced by the mice. This demonstrates that infection by Plasmodium alters both infected mosquito feeding behavior and makes infected mammals appears more attractive to uninfected mosquitos. We will discuss how these parasite strategies could affect transmission in endemic regions and how they could be used in the fight against these Machiavellian parasites.

11 Of mice, rats, cats, leopards and chimpanzees

Several classes of parasites can modify the behaviors of their hosts to improve their transmission. This ability is particularly seen with parasites transmitted through the food chain, as the immature parasite infecting an intermediate host must be eaten by a predatory definitive host to complete its life cycle. Toxoplasma falls into that class of food chain transmitted parasites.

Although the parasite can infect any mammal, by infecting rodents these pathogens get transmitted to intermediate hosts to be eaten by members of the cat family, the definitive hosts. Since felids are needed to complete its life cycle, the parasite might have a strong selective pressure to modify the behavior of rodents. The first paper by Berdoy et al. measures the overall response of uninfected and infected rats to cat and rabbit odors, providing evidence that rats infected with Toxoplasma increase their risk of cat predation.

This behavioral modification might be explained by the second paper of this session in which Prandovsky et al. show that upon infection of mammalian neurons by Toxoplasma, a significant increase in dopamine metabolism is observed. Dopamine dysregulation contributes to serious behavioral modifications in rodents and could explain the increase of cat predation.

Finally, we will analyze a third paper in which the authors measured the potential host modifications in our closest relative: The chimpanzee. These primates are still predated by a feline, the leopard, and the study by Poirotte et al. provides evidence that chimpanzees infected with Toxoplasma lose their innate aversion towards the presence of the feline. We will discuss the impact of the host manipulation by Toxoplasma and the statistical tools that were used to test the hypothesis of the authors.

Feedback and Evaluation

In this last session, we will spend some time to discuss and critique the course itself and ask the students to complete the course evaluation.

12 Oral presentations

Each student will deliver their final assignment, a 10–15 minute oral presentation that they have prepared as described in the Assignments section.