Monday, February 5, 2018

Slime Mold Blog Post

by EG


From here
“Slime mold 2020!” “Elect Physarum polycephalum to make America great again!” Have you ever found yourself deeply concerned about the current leadership of the country? Have you ever been ashamed over the lack of intelligence possessed by the man sitting in the highest office of our nation? Worry no more! In this blog post, I will present a viable alternative to the current bozo who spends all of his time everywhere but Washington: the slime mold, Physarum polycephalum. P. polycephalum is a protist that has demonstrated the mastery of efficiency, the capacity to learn, and the ability to solve complex problems, all of which are qualities lacking in the current administration. If change is what you’re looking for, fear no more, Physarum polycephalum is on the scene.
You may find yourself asking, “What are protists?”. They are an immensely diverse group of organisms with only one defining characteristic: they are all classified as eukaryotic. There are many well-known organisms, like amoeba and algae, that fall under the category of protist. To be considered a member of the kingdom Eukarya, an organism must have highly organized cells with a nucleus containing genetic information and complex organelles to carry out cell processes. However, unlike human beings, protists are composed only of a single cell. With that, cell similarities virtually stop.
A simple description is that protists are anything that cannot be classified as animal, plant, or fungus. As you can imagine, that definition encompasses a wide range of diversity. For nutrition, protists can either use light energy via photosynthesis, similar to the process used by plants, or they can use energy in the form of nutrients “eaten” by the cell. In most cases, protists also have organelles called mitochondria that, if anyone remembers high school biology, are the powerhouses of cells, and act as a currency exchange. This exchange turns nutrients into the cell recognized currency of ATP. Even so, some protists lack mitochondria, but this typically only occurs in anoxic conditions. In which case, the organism uses a hydrogenosome that functions in a similar way to mitochondria. In addition, the diversity seen in this taxa carries over to reproduction. Asexual reproduction via budding, is most common, but sexual reproductive cycles have been observed, they just remain poorly studied.
As a member of the very diverse group described above, Physarum polycephalum has a diverse range of characteristics. As previously noted, it is a slime mold, so named because it leaves behind a thick mat of extracellular slime when it moves. It is a pretty yellow color that eats fungal spores, bacteria, and various other microorganisms. Already it sounds better than the orange cheeto currently entrenched in the White House, right? It spends most of its life in a plasmodium form. While in this form, the microbe eats, divides by mitosis, and remains largely sedentary. When nutrients become rare, the plasmodium goes into starvation mode, during which motility increases rapidly and mitotic division decreases. If the plasmodium is unable to locate nutrients, a majority of the organism will desiccate and spore formation will initiate. These spores are formed in structures called sporangia, which are designed to burst when agitated, thus spreading the spores to new, hopefully nutrient-abundant, environments. The spores can then survive for an indefinite amount of time until nutrients become available. This method of reproduction ensures the survival of the species (Guttes). While the mold is good at surviving, it has also been shown to be extremely smart through rigorous testing.
One good quality to have if you’re attempting to run a country is intelligence. Otherwise, things might get out of hand and you could end up needing to be babysat by your co-workers. A key requirement for intelligence is the ability to learn and change behavior based on prior outcomes, and surprisingly, Physarum polycephalum has shown this quality. The ability to learn has been traditionally regarded as a trait found only in organisms with higher order thinking and even then, not in every case. One specific type of learning is habituation: the diminishing physical or emotional response to a repeated stimulus. Human brains are great at demonstrating habituation. For example, the initial shock of learning that a presidential candidate has no decorum or tact caused waves of anger, but by now, we’ve just come to expect it and there is a progressively smaller reaction as each new idiotic moment comes to pass. P. polycephalum is no different. In a groundbreaking study by Boisseau et al., the data suggested that the mold displayed habituation to an unpleasant, but not harmful, stimulus.
To obtain these results, the authors cultured the mold on an agar plate and connected the original plate to a second plate containing a preferred food source by building an agar “bridge.” On average, it took the mold two hours to move from the first plate to the second plate using the bridge. Once the average time it took to move had been established, the agar bridge was filled with quinine and/or caffeine, both of which are compounds that irritate the mold, but do no harm to it. The same mold that initially took two hours to cross the pure agar bridge took over twice as long to cross the quinine/caffeine bridge, and even so, crossed the bridge in a thin line in an attempt to avoid the irritant. However, when the experiment was repeated multiple times with the same mold cultures, bridge-crossing times significantly decreased, and eventually matched the initial pure agar bridge-crossing times. These results suggest that the slime mold learned that even though the quinine and caffeine were unpleasant, the compounds weren’t going to hurt it and could be crossed to reach a food source.
Another requisite trait to have as the leader of a country is the ability to think on your feet and solve problems. These slime molds also have the capacity to problem solve using their past experiences as a reference point, if being able to learn and share knowledge wasn’t cool enough. Problem solving is another skillset that has previously been relegated to higher functioning organisms only. Again, human beings are generally pretty good at problem solving. We can easily adjust our environments to be more favorable for our survival; if we’re cold, we put on a sweater, or if we’re angry, we spew vitriol onto the internet via Twitt-- wait, never mind, that doesn’t solve anything. However, this mold is out to prove that instead of throwing a temper tantrum when things don’t go as planned, it can calmly solve the problem to get the desired outcome.
In an experiment done by Reid et al., it was shown that this organism relies on spatial memory to solve a U-shaped trap, a problem often used to test autonomous navigational ability in robotics. The way the test was set up removed the ability of the slime mold to rely on chemoattractant gradients to guide its movement towards food, which is what it typically uses to navigate. If you remember to a few paragraphs back, you’ll recall that the mold leaves behind a thick layer of extracellular slime as it moves. The authors noticed that when the organism is foraging for nutrients, it strongly avoids that extracellular slime. However, when all of the new territory has been explored, the plasmodium loses its aversion to the slime and will cease its avoidant behavior. The authors concluded that the mold was “choosing” to not go where the slime was present because it signaled that area had already been explored.
From here
Using that information, the authors modified the U shaped trap for their own purposes. The U-shaped trap put a desirable food source behind a U-shaped barrier. If the mold tried to rely on classic chemoattractant signals, it would repeatedly run into the barrier and remain hungry. If it could problem solve and work around the barrier, it would signal that the mold successfully used its spatial memory to escape the trap. Turns out, the mold did just that. Ninety six percent of the plasmodia reached the food within the experimental time limit of 120 hours. I wonder what would happen if we placed a certain president in a U-shaped trap. Would he use his brain to get out, or just sit down and say the trap is rigged against him? I’m inclined to choose the latter-- there’s naught in the way of a brain. Not a slime mold, though! Those guys actually get things done.
If it was even still a question of who (or what) would make a better president, consider the fact that slime molds are just plain efficient. Evolution demands that every resource is put to use, so it’s just second nature for the mold. Efficiency gets it done. As a president, you need to be able to get things done in a timely manner in a way that’s beneficial to the most people. A lack of efficiency can lead to the waste of precious resources and create a plethora of miscommunication. Ring a bell for anyone? Physarum polycephalum understands this and actively seeks to maximize its efficiency because it increases the likelihood that it will survive to propagate, thus keeping it fit and able to lead the country.
Think back to the study done by Boisseau et al. where P. polycephalum became habituated to quinine and caffeine. The researchers took their experiments a step further and tested how long the organism remained habituated to the chemicals. In a shocking development, they found that the mold had a long term memory and could pass their habituation on to other “virgin” molds that had never encountered caffeine or quinine. Even when the habituated mold was mixed with virginal mold in a 1:9 ratio, the entire new fusion mold became habituated to the chemicals. Further research is required to determine by what mechanism the transfer of knowledge occurs, but in the case of leadership, do we really care about how knowledge gets passed on or just that it does? Unless you’re a certain person obsessed with emails. Then it matters; it matters very much.
Even more proof of the mold’s capacity for efficiency was demonstrated by researchers in Tokyo. Through their research, they showed that Physarum polycephalum is more efficient than some of the world’s leading engineers. The mold will always form the shortest pathway between two food sources to optimize resources. To test this mechanism, researchers set up food on agar to mimic the major city centers of the greater Tokyo area and allowed the mold to grow. The connective filaments of the mold matched the public transportation, including roads and subways, present in and around Tokyo at a high rate, and was found to be even more efficient than the current city design (Wantanabe).
From here
At this point, there should be no doubt about who or what would make the best supposed leader of the free world. Slime molds, members of the domain Eukarya and kingdom Protist, have got it all. They’ve proven to have the capacity to learn, demonstrated a high intelligence, and are masters of efficiency. They doesn’t throw temper tantrums, unleash juvenile tweetstorms, or waste billions of taxpayer dollars. Slime molds are pretty to look at, just want to live their lives, and for you to do the same. Slime molds aren’t an ugly orange color, they don’t have any hands over which to obsess about their size, and would never engage in “locker room talk.” I don’t know about you, but I personally welcome a new era with the supreme slime mold overlord, Physarum polycephalum.


References

Guttes, E., Guttes, S., and Rusch, H.P. (1961). Morphological observations on growth and differentiation of Physarum polycephalum grown in pure culture. Developmental Biology 3, 588–614.

Boisseau, R.P., Vogel, D., and Dussutour, A. (2016). Habituation in non-neural organisms: evidence from slime moulds. Proc. R. Soc. B 283, 20160446.

Reid, C.R., Latty, T., Dussutour, A., and Beekman, M. (2012). Slime mold uses an externalized spatial “memory” to navigate in complex environments. PNAS 109, 17490–17494.

Watanabe, S., Tero, A., Takamatsu, A., and Nakagaki, T. (2011). Traffic optimization in railroad networks using an algorithm mimicking an amoeba-like organism, Physarum plasmodium. Biosystems 105, 225–232.

Monday, January 29, 2018

The Death Defying Stunts of Trypanosomes

by EB

How much do you hate being sick, feeling better for a while, and then getting sick again? In the two deadly human illnesses caused by Trypanosoma brucei and Trypanosoma cruzi—African Sleeping Sickness and Chagas’ Disease, respectively—this cycle becomes a reality. These diseases are known for their unique symptoms: for example, African Sleeping Sickness causes the host to go through cycles of feeling ill and then feeling better every few weeks, while Chagas Disease there can be no symptoms after the initial infection for 20+ years [1, 2]!
Figure 1: Trypanosome. The arrow points to a Trypanosome.
African Sleeping Sickness, which is caused by T. brucei, had large outbreaks during the 1800s in rural sub-Saharan Africa and was almost eradicated in the 1960s [1]. However, there are still significant numbers of new cases of this disease, about 10,000, that occur each year [2]. In comparison, Chagas’ Disease, caused by T. cruzi, is endemic in almost all Latin American countries and is only present in the western hemisphere [3]. These diseases are very hard to treat because Trypanosomes are eukaryotes, meaning that many of the mechanisms that antibiotics use to treat other diseases are ineffective against them. Additionally, as they are eukaryotes, like us, stronger drugs that could treat them cause very bad side-effects, as the drugs can’t differentiate between a human cell and a Trypanosoma cell [4].

Additionally, Trypanosoma has unique mechanisms for evading the immune system, and therefore death. The best characterized mechanism is that of variable surface glycoprotein coats (VSG), which are proteins on the surface of cell [5]. The VSG that the Trypanosoma expresses changes throughout the course of the infection due to gene conversion and telomere exchanges during reproduction and replication of the parasite [5], please see Figure 2. Gene conversion is where a piece of ‘donor’ DNA gets placed into other chromosomes, which causes a change in the expression of the genes [6]. Telomeric exchanges are similar in that a piece of ‘donor’ DNA is inserted into another chromosome, however, in this case, the piece of DNA that is replaced is on the end of the chromosome—the telomere—instead of anywhere on the chromosome as in gene conversion.
Figure 2: VSG segments change by two major mechanisms as shown above. Gene conversion: First, a double stranded break in the DNA occurs, and DNA around the broken area is deleted, so that the cell can more easily repair it. Then, a ‘donor’ piece of DNA from a different chromosome comes into the broken area, where it is replicated as a template for the cell. This ‘donor’ piece of DNA is now present in the broken area, creating non-broken DNA once again. Telomeric VSG Conversion: During replication of the chromosomes, one end of the DNA on one chromosome ‘invades’ the telomere of another chromosome while it is being replicated. Due to this ‘invasion’, the DNA from the telomere of the first chromosome will become the DNA sequence at the telomere of the chromosome that was ‘invaded’. From here.
The cycle of feeling sick and then better that characterizes African Sleeping Sickness is caused by the immune system and the parasite being in opposition. When the patient feels ill, the parasite load in the blood is high and the immune system is attacking T. brucei. However, when the patient feels better, the parasites have simply changed their VSG thus evading the immune system for a few days until the immune system can make more ‘soldier’ cells to defend the body. Due to the chronic infections seen in these diseases and the difficulty in treatment, it is important to understand how Trypanosoma can evade the immune system. As not much is known about how they do this, research is being conducted upon two types of proteins: expressed and secreted. This research has determined that these types of protein play a large role in the ability of Trypanosoma in escaping death.

Secreted proteins are important to living organisms in regulation of protein activities and cell-cell communication. Due to this variation of function, there are several groups of proteins that include secreted proteins; however, we’ll be focusing on the proteins involved in folding and degradation, and enzymes of nucleotide metabolism [7]. Now for some more definitions. Folding is necessary for protein function—think of it like origami, if you’re trying to make a crane but instead make a crumpled ball it obviously can’t be a crane. Folding of proteins is exactly like that, except that if they aren’t folded in the correct ways they can’t perform their functions. Degradation of proteins is also necessary, as if too many proteins are floating around but not being used, energy is being wasted and potential harmful effects could occur. Think of this like garbage floating in a delicate ecosystem. If the garbage isn’t gotten rid of, the animals living there start dying or moving away. Next, Nucleotide metabolism is the degradation of extra nucleotides [7]. Finally, let’s define what a secretome is. Secretomes are defined as all the secreted proteins of an organism. Therefore, the investigation of secretome proteins was of high importance, especially because the VSG proteins are in the secretome. As stated above, VSG proteins are highly variable and switch every couple of weeks during the course of an infection. This variation of the parasite allows it to better evade the immune system because the immune system trigger that infected cells present changes, and so the B and T cells—think of as the generals of the immune system—for the new trigger must be activated and the ‘old’ B and T cells must become memory cells, so that is the trigger goes back to the ‘old’ trigger, the generals can be ready to marshal the troops to fight once again.
Figure 3: B and T Cells Broken Down.
The figure above breaks down the components of B and T cells and what they do for the immune system. B cells are divided into two types of cells—memory, which hang around and are ready to defend the body if the immune reactant re-emerges, and effector B cells which secrete antibodies as an immune response. T cells are initially divided into two types—CD4 and CD8 cells. CD4 T cells secrete compounds to activate other immune cells for help in clearing out the immune reactant, while CD8 T cells secrete cytokines to kill infected cells.
This research identified two important proteins that were involved in the folding and degradation of proteins. They were called cyclophilin A and heat shock protein. Cyclophilin A and heat shock proteins are known to regulate the immune system of mammals and to act as mediators for intercellular signaling [7]. Intercellular signaling allows nearby cells to communicate with each other, so that if one is sick of dying the other cells around it know. These functions are important for the escape of Trypanosoma from the host’s immune system, as effectively monitoring intercellular signaling and modulation of the immune system would aid the parasites in evasion of the killer immune cells. The proteins in the enzymatic nucleotide metabolism group are thought to be important to the parasites to degrade nucleotides around them to decrease inflammatory reactions of signals carried by them to the immune system and other cells [7]. These inflammatory reactions are one of the most basic defenses of our immune systems, and so if this reaction isn’t carried out precisely, it won’t be effective, and could even kill healthy cells. Based upon this evidence, it is clear that secreted proteins may have an effect upon the Trypanosoma’s escape from the host’s immune system, as the proteins found modulate the immune system, the inflammatory response, and intercellular signaling.

Before we go on talking about Trypanosoma’s death defying stunts, let’s define a few more terms. First, what are surface proteins? Surface proteins are proteins that are located on the cell membrane of a cell and are exposed to the environment around the cell. They also allow transport in and out of the cell and engage in communication between cells; they are important to look at for further applications for the creation of drugs. CD4 T cells are part of the immune system, and are a type of ‘general’. These ‘generals’ release cytokines and interleukins, which are the ‘soldiers’ of the immune system to combat the enemy, in this case Trypanosoma, through inflammatory reactions that cause death, and the activation of more ‘generals’ and ‘soldiers’.

Now that we know what these terms are, let’s continue our discussion about how Trypanosoma evade the immune system. Further research was conducted upon a group of surface proteins called Sialoglycoproteins of Trypanosoma cruzi. These It was found that the Sialoglycoproteins, specifically one called O-glycoslyated Thr/Ser/Pro-rich mucin molecules, also known as TcMuc, can cause inhibitory effects on CD4 T cells and a decrease in the production of cytokines [8]. In fact, very small TcMuc concentrations resulted in inhibition of CD4 T cell proliferation. This inhibition of one of the ‘generals’ of the immune system was not overcome with the inclusion of Interleukin-2 (IL-2) [8]. This interleukin is necessary for the activation of the CD4 general cell, which demonstrates how bad this protein is for the human immune system. As CD4 T cells are a major component of the human adaptive immune system, therefore, without CD4 T cells, an immunocompromised individual can occur. IL-2 meanwhile, is an important signal to both CD4 T cells and CD8 T cells; it is a cytokine that helps T cells to proliferate. Therefore, this one molecule on the surface of T. cruzei effectively cripples the adaptive immune system. If T cells can’t proliferate, a large response to the parasite would not be able to be formed and the immune response compromised.

Furthermore, TcMuc downregulates expression of certain cytokines, the ‘soldiers’, see Figure 3 [8]. These cytokines that are expressed at lower dosages are highly important in the immune response as they activate various immune cells, such as different varieties of CD4 T cells like TH1, TH2, and Treg, and cause systemic effects that tell the host that they are sick. These other CD4 T cells are like ‘captains’ of units that have specific functions; for example, Treg ‘captains’ molecules that regulate autoimmunity—without these cells, everyone would have at least one autoimmune disease—while TH1, and TH2 CD4 T cells participate in the immune response towards the parasite. With downregulated expression of these cytokines, the immune system has a harder job to complete in order to become activated (IL-2) and prepared to fight the parasitic infection (cytokines). Furthermore, TcMuc sialylation decreases dendritic cell function [8]. Dendritic cells are a major part of one’s innate immune system. They process immune triggers and bring them into the lymph nodes, where B and T cells mature, in an attempt to activate the appropriate adaptive cells. Therefore, if their ability to present antigen and bring it to the lymph nodes is compromised, the adaptive immune system is unable to complete their job and clear the infection.

Trypanosomes engage various mechanisms to evade the immune system of their hosts. Commonly, these mechanisms are associated with secreted proteins on the surface of the cell. Treatment for Trypanosoma sickness is complex because the drugs used also target human cells. As these organisms are eukaryotes, drug targets that do not have homologs in humans are challenging to find. The evasion mechanisms using the proteins discussed above all have homologs in humans; therefore, more research is needed on secreted and surface protein functions of Trypanosomes. This research may uncover potential drug targets that do not have homologs in humans and open new paths for treatment of African Sleeping Sickness and Chagas’ Disease.


References:
[1] Brun, R., Blum, J., Chappuis, F. and Burri, C. (2010). Human African trypanosomiasis. The Lancet, 375(9709), pp.148-159.

[2] Cdc.gov. (2017). CDC - African Trypanosomiasis. [online] Available at: https://www.cdc.gov/parasites/sleepingsickness/index.html [Accessed 27 Nov. 2017].

[3]Desforges, J. and Kirchhoff, L. (1993). American Trypanosomiasis (Chagas' Disease) -- A Tropical Disease Now in the United States. New England Journal of Medicine, 329(9), pp.639-644.

[4] Cdc.gov. (2017). CDC - African Trypanosomiasis - Resources for Health Professionals. [online] Available at: https://www.cdc.gov/parasites/sleepingsickness/health_professionals/index.html#tx [Accessed 27 Nov. 2017].

[5] Marcello, L. and Barry, J. (2007). Analysis of the VSG gene silent archive in Trypanosoma brucei reveals that mosaic gene expression is prominent in antigenic variation and is favored by archive substructure. Genome Research, 17(9), pp.1344-1352.

[6] Chen, J., Cooper, D., Chuzhanova, N., Férec, C. and Patrinos, G. (2007). Gene conversion: mechanisms, evolution and human disease. Nature Reviews Genetics, 8(10), pp.762-775.

[7] Geiger, A., Hirtz, C., Bécue, T., Bellard, E., Centeno, D., Gargani, D., … Peltier, J.-B. (2010). Exocytosis and protein secretion in TrypanosomaBMC Microbiology10, 20. http://doi.org/10.1186/1471-2180-10-20

[8] Nunes, M., Fortes, B., Silva-Filho, J., Terra-Granado, E., Santos, L., Conde, L., de Araújo Oliveira, I., Freire-de-Lima, L., Martins, M., Pinheiro, A., Takyia, C., Freire-de-Lima, C., Todeschini, A., DosReis, G. and Morrot, A. (2013). Inhibitory Effects of Trypanosoma cruzi Sialoglycoproteins on CD4+ T Cells Are Associated with Increased Susceptibility to Infection. PLoS ONE, 8(10), p.e77568.