New Thoughts on How Plasmodium Changes its Spots

Plasmodium falciparum is the leading cause of malaria in people, although there are Plasmodium species, spp., that infect virtually all the tetrapods (mammals, birds, reptiles, amphibians). As most people know, malaria is acquired from mosquito bites (Anopheles mosquitoes to be specific), because Plasmodium spp. have complex life cycles that require both an insect (mosquito) and a tetrapod host. Importantly, there is essentially no overlap between the Plasmodium spp. that cause frog, mouse, or human malaria. This host specificity is due to the inability of the parasite to survive in the wrong host as well as the general specificity mosquitoes show in source of bloodmeal, in other words mosquitoes that feed on frogs do not feed on humans.


Now in order to successfully infect a human being, P. falciparum cells must deal with the immune system. The immune system is exquisitely good at destroying foreign invaders, which is why you are alive to read this. So in order for an organism to survive within your tissues and bloodstream it must have a way (or ways) to deal with your immune system. Some organisms simply destroy important parts of the immune system, helper T-cells in the case of HIV, thereby short circuiting the entire system. However, Plasmodium spp, at least the ones that infect mammalian hosts take a different approach. They duck and weave.

Mickey: "I said duck and weave!"

From the CDC. We are dealing primarily with the bottom right blue circle. 

Much like a virus, Plasmodium spp. can only grow within an infected cell. When P. falciparum enters the human bloodstream from the mosquito, it first infects liver cells (hepatocytes). Here it divides a bunch of times and differentiates into cells called merrozoites. At this point the immune system is essentially unaware that P. falciparum parasites are growing within the body. Once parasites (merozoites) burst from the hepatocytes, they enter the bloodstream and can infect only red blood cells. This they do quite well, which leads to the production of more parasites (within the red blood cell), which burst forth from the now dead red blood cell looking for fresh red blood cells to infect. This ultimately leads to the reduction in red blood cells in your body, which lead to many of complications associated with malaria.

Marti figure showing Plasmodium within a PV.

What is interesting is that while growing within the red blood cell, the Plasmodium cell is actually growing within a bag inside the red blood cell called the parasitophorus vacuole (PV) not in the cytoplasm (this figure from a review by Matthias Marti et al shows a real life and a cartoon version of PVs within a red blood cell). So the immediate environment from the Plasmodium's perspective is the PV (the yellow area in the cartoon). However, the source of Plasmodium's nutrient come from the cytoplasm of the red blood cell (the red area in the cartoon). As you might expect, Plasmodium secretes proteins into the yellow area that enter the PV membrane and are used to take up nutrients from the red blood cell cytoplasm. (Proteins within the Plasmodium plasma membrane can then move nutrients from the PV into the cytoplasm of the Plasmodium cell). Damn, that's complicated. You might want to reread this paragraph again.


Now everything is well and good from Plasmodium's perspecitve, it can get enough food. However, it still interacts with the more distal environment, the bloodstream, which means Plasmodium needs to make proteins that enter the PV, cross the PV membrane enter the red blood cell cytoplasm, and then enter the plasma membrane of the red blood cell to be able to see what's going on in the bloodstream!!!


The major protein that actually does this is called the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1 for short). For the record, an erythrocyte is a fancy word for red blood cell. So PfEMP1 is out there interacting with the bloodstream doing all the things it needs to do, which is primarily interacting with other host (your) cells.


Sounds like everything is kosher for Plasmodium right? Wrong. Once this protein, which is made by a foreign invader, is exposed on the bloodstream, your immune system can see it. You may recall that your immune system kicks ass, so once it sees this PfEMP1 protein hanging out, it will ramp up the defenses and kill the red blood cell (including the growing Plasmodium cells within it). YAY! for you, boo for Plasmodium. It can also kill any RBCs infected with Plasmodium expressing PfEMP1. 


Obviously, malaria wouldn't kill 3 million people a year if it let the immune system kick its ass. Nope Plasmodium learned long ago (yes Im anthropomorphizing) that sticking PfEMP1 out of the red blood cell is essential yet lethal. To overcome the lethality (from your immune system), Plasmodium encodes ~60 slightly different PfEMP1 proteins. As the immune system ramps up its defenses against one specific PfEMP1, a small subset of Plasmodium cells express a different PfEMP1 protein. The immune system kills off the red blood cells infected with the Plasmodium expressing the original PfEMP1 protein, but is completely unaware of the PfEMP1 protein being expressed. These new Plasmodium grow like mad, killing more red blood cells, until the immune system resets and notices the new PfEMP1 protein and the system repeats. All the while you are losing more and more red blood cells and your immune system is becoming less efficient because you are tired and weak. Ultimately you will die or your immune system will finally gain an edge (6-12 months later) and you recover.


This process of swapping proteins to avoid the immune system is referred to as antigenic variation, antigenic switching, phase variation, phenotypic switching, etc. Probably the best known example of this is the Trypanosomes (the topic for another post).

Trypanosome

A Plasmodium cell is thought to only express a single form of PfEMP1 on the red blood cell. Upon infection of a new red blood cell, the specific form of PfEMP1 to be expressed can change in a very small subset of cells, but still only a single form of PfEMP1 is expressed. This is the "duck and weave" strategy mentioned above. However, a recent report in PLoS Pathogens suggests that things may not be so clear cut as one type of PfEMP1/red blood cell. 

Adapted from Figure 1 of Joergensen et al.

The authors grew P. falciparum cells that express a specific PfEMP1 (PFD1235w) in culture for a short time and then looked for expression of the original PFD1235w PfEMP1 as well as a different PfEMP1, PF11_0008. Lo and behold, the authors found infected red blood cells that had both PfEMP1 proteins expressed! To walk you through this, the authors used antibodies specific to either PFD1235w or PF11_0008, these antibodies bind to their respective PfEMP1. You can then add a fluorescently labeled antibody that only binds to the original antibody used in the first step. This gives the green and red colors. You will note that in rows A and B only green or red staining is observed. These are controls showing that this approach works and is specific (the antibodies are not cross-reacting) Thus, the red blood cells infected with  P. falciparum expressing just one or the other PfEMP1 do not have any crossreactivity with the antibodies being used for detection. Row C is the money row. This red blood cell is infected with P. falciparum expressing both PFD1235w and PF11_0008 and thus is both red and green. This result suggests that at least when grown in culture, P. falciparum can express more than a single PfEMP1 within the red blood cell. The authors include a ton more controls, in fact most of the paper is controls to demonstrate this result is real.

Adapted from Figure 6 of Joergensen et al.

So why is this important? First, the dogma, and I use this term loosely, was one and only one PfEMP1 form can be expressed in an infected red blood cell at any time. This paper suggests that we should rethink the strength of this original idea. Second, PfEMP1 is important to interact with additional host cells, primarily the endothelial cells lining the blood vessels. This interaction allows P. falciparum-infected red blood cells to adhere to endothelial cells (which can help lead to things like cerebral malaria. Third, the results reminded me of something I thought was extremely interesting. I noted that PfEMP1 interacts with endothelial cells. Well, it does this by binding to specific proteins found on endothelial cells. Proteins like ICAM-1 and CD31 (PECAM1). The authors actually looked into this issue and found that infected red blood cells expressing both PFD1235w and PF11_0008 PfEMP1s bind better to endothelial cells (see figure). The first two bars (black and light gray) are infected red blood cells expressing a single PfEMP1 and the third bar (darker gray) is infected red blood cells expressing both PfEMP1s. The fourth bar (another light gray) is an extremely smart control. This sample is basically a mix of the samples used in for the first two bars. This shows that there is no in trans effects between red blood cells. Finally, the fifth bar is yet another control that you can read up on in the paper if you are interested. So why is this "extremely interesting" to me? Well when thinking about this paper, I recalled some immunology I had eons ago and reread several years back dealing with these same endothelial cell proteins.

Adapted from Immunobiology Figure 2.36

See neutrophils, critical cells of innate immunity, travel through the bloodstream. If you have an infection in your arm, your cells send signals to the endothelial cells to get the neutrophils over stat! (Kind of like calling 911.) Endothelial cells express ICAM-1 and other molecules that help the neutrophils pull over out of traffic and leave the bloodstream at the appropriate places where they can then take a bite out of crime kill the pathogens. Here is a clear example of evolution using the same strategy to accomplish some function. These endothelial cell molecules play critical functions in your body, such as ICAM-1 to stop neutrophils flying through the bloodstream. For Plasmodium to survive it needs to stop, or at least slow down while in the bloodstream. Why not simply use to tools already in hand at co-opt the system used for neutrophils?


Finally, it is quite simple to conclude that more binding is better and therefore infected red blood cells must frequently express distinct PfEMP1s. However, this may not be true. First, there is much evidence that infected red blood cells only express a single PfEMP1 type at a time. In fact, if different PfEMP1s were expressed at the same time, the immune system would be able to recognize them, which would more or less cause Plasmodium to duck into the punch. Second, it is unclear if this actually happens in the host or is an issue with laboratory culturing. Regardless, this paper clearly suggests new questions, which heretofore would not have been asked because it was well known that Plasmodium-infected red blood cells only express a single type of PfEMP1.

Joergensen, L., Bengtsson, D., Bengtsson, A., Ronander, E., Berger, S., Turner, L., Dalgaard, M., Cham, G., Victor, M., Lavstsen, T., Theander, T., Arnot, D., & Jensen, A. (2010). Surface Co-Expression of Two Different PfEMP1 Antigens on Single Plasmodium falciparum-Infected Erythrocytes Facilitates Binding to ICAM1 and PECAM1 PLoS Pathogens, 6 (9) DOI: 10.1371/journal.ppat.1001083