Lecture Summaries

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.

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.

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.

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.

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.

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