Toxoplasma and the Invisibility Cloak

Sometimes the best disguise is not to be seen at all. This is the strategy of the intracellular parasite, Toxoplasma, which as it invades your cells, it actively modifies its compartment-of-residence so that it can’t be recognised, not even by the cell in which it is living!

Ever heard of the strange rule that pregnant women should never clean cat litter boxes? Well I hadn’t either, that is until I took my first parasitology class. In any case, it’s for fear of the development of toxoplasmosis, an infection by the protozoan parasite Toxoplasma gondii, during pregnancy that gives expecting mothers reprieve of at least one cleaning duty. (In defence of our feline friends, though it is true that cats harbour the sexual stages of this parasite, the majority of Toxoplasma infections come from eating poorly cooked meat… just ask the French, as historically upwards of 90% of France’s population tested positive for having had contact with this parasite irrespective of their experience in cleaning litter boxes.[1]) It is specifically infection *during* pregnancy that can result with miscarriage. Which is why my Spanish supervisor tells stories of her married friends who express relief upon discovery that they are infected *before* they start “doing it” in earnest.

Toxoplasma is an obligate intracellular parasite – it needs to invade host cells in order to survive. Incredibly, once inside, the parasite appears to be shut out from the outside world: surveillance cells of the immune system cannot detect the parasite as the infected cell fails to put out any typical warning signs. It seems that Toxoplasma either somehow interferes with the cell’s attempt to raise its little white flag (“Help! Something’s got me!”), or has developed the means to hide from its host – donning an invisibility cloak, if you will. A paper from the Sibley group at Washington University provides evidence for the latter in an intriguing paper as published in 1999 in the Journal of Experimental Medicine (I still have yet to determine what “experimental medicine” exactly is… it’s definitely not about mixing drugs of choice, I can tell you that much).[2]

It is known that Toxoplasma invades cells via an active process (in contrast to the bacteria that causes tuberculosis, which appears to allow itself to be eaten by the cell). During this invasion process, the parasite will push its way into the plasma membrane, donning the membrane as a coat so that it can live in its own compartment within the cell. This compartment, and all other compartments which hold parasites intracellularly, is known as the parasitophorous vacuole. Unlike all other vacuoles inside the cell, which can be controlled by the cell with respect to their movement and activity, other researchers have noticed that the parasitophorous vacuole of Toxoplasma resists such manipulation. Most notably, the vacuole doesn’t appear to fuse with lysosomes, a process used by the cell to digest both incoming nutrients as well as to deliver an insulting pathogen to a timely and deserved death.[3]

Toxoplasma is its own interior decorator

What might be going on? How is the parasite able to avoid recognition, especially if it is wrapping itself with the cell’s own membrane? The Sibley group had several hints. First, it had been observed using high-powered microscopy techniques that the PV of Toxoplasma looked void of proteins that crossed the membrane, despite the fact that the cell’s plasma membrane (from which the PV was made, remember) is chocked full of these proteins. Second, that Toxoplasma, in its active invasion process, creates a tight constriction on the cell surface called the “moving junction.” It was therefore hypothesised that Toxoplasma uses this moving junction as a sieve – a point where it could sort out which proteins from the plasma membrane it would like to include and which to exclude from its soon-to-be home (if only we could all be so lucky to have such selective choice over our furnishings and décor). From the view of the parasite, transmembrane proteins would be a dead give away because they would extend into the cell cytoplasm where they could be recognised by the cell. Toxoplasma’s ingenious solution? Just get rid of all those conspicuous proteins from its PV in the first place! And that is exactly what the Sibley group set out to prove in this paper.

The general experimental setup involved adding parasites to a layer of cells that had adhered to the bottom of a petri dish. After only 5 minutes, the dish was washed, thereby removing any free-floating parasites that had failed to begin the invasion process (in other words, those parasites better act QUICK!). The parasite-invaded cells were then fixed and stained for observation under the microscope. In some cases, rather than staining after invasion, the cells were stained before invasion. Zymosan (a component of yeast cell walls) was used as a control, as it is already understood that cells eat zymosan like it’s going out of style (through a process called endocytosis … I just think that turn of phrase is hilarious and had to use it in this entry somewhere).

Yes to Lipids! No to Proteins!


© The Rockefeller University Press
In the first figure of the paper, the cell surface had been pre-stained for surface proteins (middle panels) and for lipids in the plasma membrane (right panels). First look at how the cell ingests zymosan in the bottom row: the zymosan particle is surrounded both by the membrane proteins and lipids, indicating that its vacuole is composed of both. Contrast that to when Toxoplasma invades (top row). Though you can see that the Toxoplasma vacuole contains the lipids from the plasma membrane, in the space where you expect to see the surface proteins is an empty void: no surface proteins make it to the vacuole. Somehow, the parasite has prevented the inclusion of surface proteins in its parasitophorous vacuole.

G.P.I.-want-you


© The Rockefeller University Press
In a similar manner, by using antibodies that recognise calveolin, Sca-1 and CD55, the Sibley group shows that Toxoplasma excludes proteins that are involved in endocytosis (calveolin, in the bottom row), while includes proteins that are not transmembrane but rather are attached to lipids on the cell surface (these proteins are “GPI-anchored,” or attached to the GPI lipids in the plasma membrane, as seen in both the top and middle rows). Thus it seems that the parasite will allow lipids and GPI-anchored proteins into its vacuoles, but not proteins that cross the membrane or are found on the internal side of the membrane.

Three Faces of ICAM-1


© The Rockefeller University Press
This seemed to indicate an overall trend: that molecules with interior parts were excluded. However as no one knows the structure for most of the molecules that have been discussed up until now, one could argue that perhaps this selection process was happening because some surface molecules were bigger than others. Thus in order to weed out this criticism, the Sibley group used a very clever genetic technique. They found cells that did not express a specific surface protein, in this case ICAM-1. Then in three different groups of cells, they inserted into each group a gene which encoded for a different form of the ICAM-1 molecule: one had the normal form that you would see in a typical human cell, transmembrane domain and all; another they instead attached the ICAM-1 to a GPI molecule thus making it “GPI-anchored;” and in the third, they removed the end of the gene that encoded for the part of the protein that extended into the cell interior (thereby removing its “intracellular tail”). They then infected these three different cell populations with Toxoplasma and observed to whether the different forms of ICAM-1 were included in the membrane. In setting up these three parallel groups of cells, a difference in how the forms of ICAM-1 molecules act could be attributed directly to their mutated tails and not to their extracellular size. And as one might have predicted based on the previous results, the ICAM-1 with the transmembrane portion was excluded (top panels) while those that were GPI-anchored or lacking their intracellular tails were included.

So what's next?

Though the molecular mechanism behind how this process works has yet to be figured out, the fact that the parasite has the means to determine how its intracellular niche will look has huge implications as to how one begins to design therapies in order to inhibit the growth of or kill the parasite. For example, as the parasite is able to prevent the fusion of other vacuoles to its home, then the cell cannot deliver its toxic enzymes to attack. Thus, using drugs to enhance those killing enzymes would be futile. At the same time, once the molecular mechanism is defined, then it will open the doors for research into how we might exploit or prevent this invasion process.

So. Congratulations. You just read your first science paper.

References:

1. Toxoplasma gondii. (2006, June 14). In Wikipedia, The Free Encyclopedia. Retrieved 01:36, June 20, 2006, from http://en.wikipedia.org/wiki/Toxoplasma_gondii
   
2. D.G. Mordue et al. (1999). Invasion by Toxoplasma gondii establishes a moving junction that selectively excludes host cell plasma membrane proteins on the basis of their membrane anchoring. J Exp Med 190:1783-92.
   
3. D.G. Mordue et al. (1999). Toxoplasma gondii resides in a vacuole that avoids fusion with host cell endocytic and exocytic vesicular trafficking pathways. Exp Parasitol 92:87-99.