The Hitchhiker’s Guide to the Amoeba

By OS

                  Most people enjoy traveling and want to experience different parts of the world. Some people, like Todd, even have it on their bucket lists to visit every country. Traveling comes with many risks though, and one major concern for travelers is disease. Unfortunately Todd discovered this for himself when he visited India. Shortly after Todd arrived in India, he went out to an unsanitary restaurant because it was the only one nearby and he did not feel like going shopping on his first night there. While there, he noticed that the water didn’t look completely clean. He figured that water in India just looked like that, so he drank it anyway. In doing so, he exposed himself to the amoeba Entamoeba histolytica, which is the pathogen responsible for amebiasis. Amebiasis causes disease when its cysts are ingested and is responsible for an estimated 100,000 deaths each year (1). This disease is common in areas with poor sanitation, including Central America, South America, Africa, and parts of Asia, so Todd should have been more careful (2). By staying away from unsanitary restaurants and not drinking potentially unsafe water, Todd could have avoided being infected by this parasite.

                  Todd didn’t realize it for a while, but his body was under attack. The water he drank carried E. histolytica cysts down his throat, through his stomach, and into his small intestine (figure 1). Once the cysts reached his small intestine, the parasite escaped its cyst and replicated asexually three times, forming eight trophozoites (3). Trophozoites are the motile form of the parasite that feeds on the host using porins and proteolytic enzymes. The trophozoites traveled to his large intestine and adhered to its mucosal lining. Here they degraded the layer of protective mucous and attacked the epithelial cells of the large intestine. This was followed by the release of porins, which caused some of Todd’s cells to lyse. The proteolytic enzymes were then released to partially degrade the ruptured cells. Finally, the amoeba engulfed the dead cells and used them as a food source. The parasite was literally eating him alive! The death of Todd’s cells and inflammation of his colon caused him to experience some of the symptoms associated with amebiasis, including pain and diarrhea.  The symptoms began to appear five days after infection. At this point, Todd knew something was wrong, but he figured it was just food poisoning and that it would be done shortly. He thought his immune system would fight off the infection, but he was wrong.

Figure 1. Life cycle of E. histolytica.   

                  Todd’s complement, which is part of his innate immune system, could not clear the infection. E. histolytica is resistant to the lysis that the complement system normally uses to kill infections (3). Additionally, some of his IgA antibodies were unable to help fight the disease. Antibodies normally bind pathogens and either block further infection or help immune cells destroy the pathogens. However, E. histolytica is able to quickly degrade IgA, allowing for its self-preservation. The parasite also prevented Todd’s T cells from functioning properly. T cells are immune cells that normally help eliminate specific pathogens. However, Todd’s T cells were unable to clear the infection because the parasites were secreting proteins to decrease the T cell activity (4). Todd’s other immune responses were insufficient to kill all of the parasites, so his body could not clear the amebiasis on its own.

The trophozoites kept replicating asexually and continued attacking his cells. Some trophozoites formed into cysts, which were excreted in his feces and waited until they found another person to infect (3). After another week, his amebiasis became more severe and the trophozoites reached his bloodstream. From here, they could have traveled to any tissue in his body, including his lungs or brain. Wherever the parasites travel, they kill cells, so travel to other areas of Todd’s body may have caused serious damage. Todd was lucky that the E. histolytica cells did not infect his brain or lungs. Infection of these areas is rare, but can be very serious. However, they did travel to his liver. When the E. histolytica infection reached Todd’s liver, it killed some of his liver cells and formed abscesses, which are clusters of dead cells, immune cells, and parasitic cells (5). As his liver was infected, Todd started experiencing fever, coughing, and other flu-like symptoms, as well as intense pain. When he noticed his symptoms weren’t getting better, he finally went to see the doctor.

The doctor asked about his ailment and determined the cause may have been due to an ingested parasite. He ordered a blood test in order to precisely determine the cause. The blood was sent to be analyzed and an enzyme-linked immunosorbent assay (ELISA) was performed. This ELISA was done in order to see if Todd had antibodies against several parasites, including E. histolytica (6). This can show if someone is infected because the human body normally creates antibodies to fight against infectious pathogens. The presence of antibodies specific to a pathogen shows that the body is actively fighting against the pathogen. The test showed that Todd’s blood did contain antibodies to E. histolytica, so the doctor concluded that amebiasis was the cause of his symptoms. Because of this, the doctor was able to prescribe him metronidazole, which is an amebicidal drug that is very effective at killing E. histolytica (7). This helped Todd to finally clear the infection.

Fortunately for Todd, his infection had not spread too far and it went away after treatment. However, being more careful could have prevented him from being infected at all. It is always important to be careful about what you eat and drink, but paying attention can mean the difference between life and death for immunocompromised or other vulnerable people. People with AIDS, SCID, or other immune deficiencies need to pay special attention because amebiasis can be very severe for people with weak immune systems. Todd plans to continue traveling the world, but now he knows to be more careful and take precautions to stay healthy. Amebiasis, as well as many other diseases, can be prevented by taking the necessary precautions when living in or traveling to areas without proper sanitation. In addition to avoiding consumption of potentially unsafe food and water, amebiasis can be prevented by washing your hands often, keeping raw foods away from cooked foods, and ensuring meat is thoroughly cooked. By spreading this information, hopefully the prevalence of amebiasis can be reduced.

Bibliography

1.              Ali IKM, Clark CG, Petri Jr WA. 2008. Molecular epidemiology of amebiasis. Infect Genet Evol. 8:698-707

2.              Wertheim HFL, Horby P, Woodall JP. 2012. Atlas of Human Infectious Diseases. Wiley-Blackwell, Chichester

3.              Ravdin JI. 1995. Amebiasis. Clin Infect Dis. 20:1453-1464

4.              Salata RA, Martinez-Palomo A, Canales L, Murray HW, Trevino N, Ravdin JI. 1990. Suppression of T-Lymphocyte Responses to Entamoeba histolytica Antigen by Immune Sera. Infect Immun. 58:3941-3946

5.              Espinosa-Cantellano M, Martinez-Palomo A. 2000. Pathogenesis of Intestinal Amebiasis: From Molecules to Disease. Clin Microbiol Rev. 13:318-331

6.              Tanyuksel M, Petri Jr WA. 2003. Laboratory Diagnosis of Amebiasis. Clin Microbiol Rev. 16:713-729

7.              Lasserre R. 1979. Treatment of Amebiasis. Phil J Microbiol Infect Dis. 8:1-6.

Stealing the Ability to Photosynthesize and Endosymbiotic Theory

by AS

Have you ever wondered whether we could use genetic engineering to make an organism capable of photosynthesis, or maybe just how plants gained the ability in the first place? Well, studying the unique food chain of Dinophysis acuminata is helping to provide answers for questions like these. Dinophysis acuminata is a unicellular marine plankton that feeds on a ciliate plankton called Myrionecta rubra, which in turn feeds on photosynthetic algae like Geminigera cryophila. What makes this food chain interesting is all three of these species perform photosynthesis, but the two predators do it by using the cellular machinery of their prey. That is to say, rather than consuming the whole of their prey, they conserve the chloroplasts and even the genetic information necessary for chloroplast maintenance so they can perform photosynthesis. However, before we dive into the deep end of life as a plankton in the ocean, we need to discuss endosymbiotic theory. Endosymbiotic theory will reveal why this food chain adds to our understanding of evolution and what genes are necessary for photosynthesis.

To begin, cells are divided into two groups, prokaryotes and eukaryotes. All cells possess a plasma membrane dividing them from the environment, but eukaryotes are known for having internal membranes as well. These internal membranes segregate portions of the cell into compartments, called organelles, where specialized cellular functions occur. One of these organelles, the chloroplast, is what allows photosynthetic organisms to capture energy from light and store it in the form of carbohydrates, like glucose.

It’s thought chloroplasts evolved through endosymbiosis, whereby one cell, known as an endosymbiont, lives within another (Chan & Bhattacharya, 2010). The idea is a unicellular predator engulfs a cell that can photosynthesize and, instead of digesting its prey, the predator keeps the prey within it. This allows the predator, which is now the host, to feed off sugars generated by the engulfed photosynthetic cell for an extended period of time. This arrangement can also benefit the endosymbiont in numerous ways. The most obvious advantage being it is now protected from other less accommodating predators. In addition, the endosymbiont is now shielded from some environmental stresses by the host’s plasma membrane, such as harsh chemicals.

Over time, and many generations, the endosymbiont’s genome shrinks and populations of them start getting passed from parent host cells directly to the host cell’s progeny. This is opposed to progeny needing to find and engulf their own endosymbionts. Genes are transferred from the endosymbiont’s genome to the host’s with astounding frequency while other, no longer essential, genes are simply lost (Timmis et al., 2004). At a certain point, the once free-living endosymbiont no longer contains within its genome the genes necessary to survive on its own, because these genes have been transferred to the host nucleus. At this point the endosymbiont has been reduced to an organelle and has become a part of the host, rather than a partner.

Most of the endosymbiont’s genome shrinkage is believed to occur soon after the endosymbiont begins living within its host. However, keep in mind that is ‘soon’ on an evolutionary timescale, which implies millions of years. It’s been found gene transfer from organelle to host nucleus is an ongoing process and, surprisingly, many of the protein products encoded by the transferred genes are not targeted back to the organelle those genes originated from (Archibald, 2005). For instance, one study concluded about 18% of genes within the genome of the plant Arabidopsis thaliana originated from cyanobacterial endosymbionts, and less than half of the protein products of those genes were targeted to chloroplasts (Archibald, 2005). This implies gene transfer from endosymbionts has played a large role in shaping the evolution of higher eukaryotes. Since many of these transferred genes don’t associate directly with the chloroplasts they originated from anymore, this influence likely goes beyond just contributing genes involved with maintaining organelles and their cellular processes. That’s fascinating, and implies the acquisition of permanent chloroplasts was a monumental step in the evolution of eukaryotes for reasons beyond the obvious; they provided the basis for multicellular photosynthetic organisms.

Generally, it’s agreed this critical step in the evolution of eukaryotes only occurred once and all chloroplasts evolved from a single photosynthetic cyanobacterium that formed an endosymbiotic relationship with a common ancestor of all photosynthetic eukaryotes (Archibald, 2005). This is called the primary endosymbiotic origin of chloroplasts. The problem with studying this process, and the effects it’s had on eukaryotic evolution, is these events happened billions of years ago (Archibald, 2005). However, there is another level of this process we can study because it’s still ongoing today.

Secondary endosymbiosis begins with a non-photosynthetic eukaryote engulfing a eukaryote that contains some method of photosynthesizing; whether it be endosymbiotic bacteria, chloroplasts, or something in between. The predator then digests the engulfed eukaryote but keeps the photosynthesizing apparatus for itself. The number of times secondary endosymbiosis has resulted in new species with permanent chloroplasts is still debated, but there is strong evidence that it has occurred at least three times (Archibald, 2005).

Now the process of permanently acquiring chloroplasts via secondary endosymbiosis is slightly different from that undergone during the primary endosymbiotic origin. Chloroplasts no longer contain all of the genes necessary for photosynthesis or self-maintenance (Archibald, 2005, Johnson et al., 2007). Remember, large portions of the organelle’s genome were transferred to the host nucleus, and the host was just digested. This means stolen chloroplasts eventually break down and stop producing energy for their new host. In order to extend the lifetime of their stolen goods, the new host must acquire the missing genes from other eukaryotes that already keep a stable population of chloroplasts.

An excellent example of secondary endosymbiosis is the food chain of D. acuminata discussed earlier. Starting at the bottom, M. rubra engulfs algae and digests them, but keeps their chloroplasts and nuclei. Now the cool thing is the nuclei of the digested algae accumulate within M. rubra and remain transcriptionally active. Transcription is the process of turning genes into mRNA, which is translated into the proteins that mediate cellular processes, like photosynthesis. So the stolen algal nuclei continue to produce the mRNA that encode the proteins needed for maintaining chloroplasts and driving photosynthesis. However, the nuclei do have a 10-day half-life within M. rubra so it needs to feed continuously to maintain its stolen chloroplasts. (Johnson et al., 2007)

The journey of our pilfered chloroplasts doesn’t stop there however. D. acuminata feeds off M. rubra and keeps the now twice appropriated chloroplasts for itself. Unlike M. rubra, D. acuminata does not keep the original host nuclei. Yet, D. acuminata can keep their chloroplasts for months without feeding. This is possible because D. acuminata has somehow acquired several genes from algae that regulate and maintain chloroplasts. Five such genes have been identified. What makes this even more interesting is only one of them originates from a cryophyte, which are photosynthetic organisms that can live on snow or ice, such as the original owner of the chloroplasts G. cryophila (Wisecaver & Hackett, 2011). You would expect the new host to acquire the necessary genes from the original host, its prey, and this has been found to be the case with other dinoflagellates that steal their chloroplasts like D. acuminata. However, the other 4 identified genes came from algal lineages different from G. cryophila. D. acuminata obtained these algal genes through some form of horizontal gene transfer (HGT). HGT is simply defined as the transfer of genetic material between organisms that occurs through routes other than parent to offspring, which is vertical gene transfer. For example, the transfer of genes from endosymbiont to host we’ve been discussing is a form of HGT.

Thus, this food chain offers two very different approaches to maintaining chloroplasts acquired from prey. M. rubra’s method of stealing whole nuclei has never been observed before, and therefore represents a unique research opportunity. Also, since M. rubra collects the nuclei of its prey, they are frequently exposed to the genes necessary for maintaining chloroplasts permanently. Many researchers have suggested M. rubra is in the process of taking these genes into its genome and permanently acquiring chloroplasts (Johnson et al., 2006).

Meanwhile, D. acuminata employs a more haphazard approach to endosymbiosis. Since it goes through M. rubra as an intermediary for its chloroplasts, D. acuminata is exposed to the genome of the original host less frequently. Some have postulated this is why most of D. acuminata’s genes involved with chloroplast maintenance originate from sources other than the original host. It simply doesn’t have access to the original host’s genome very often so the opportunity for HGT doesn’t arise. The actual mechanisms of HGT aren’t well understood in eukaryotes though, and D. acuminata could serve as a model for further investigation concerning eukaryotic HGT as well as secondary endosymbiosis (Zhaxybayeva & Doolittle, 2011).

For these reasons both M. rubra and D. acuminata are amazing organisms that may very well provide insights into how the process of endosymbiosis produced modern eukaryotes and continues to influence their evolution today. In addition, by observing the process of HGT in these organisms and the impact of specific genes, we could determine the specific gene set necessary to permanently sustain chloroplasts. Genetically engineering photosynthetic organisms may seem straight out of sci-fi, but with a list of necessary genes and modern genome editing tools it may not be too far away. However, don’t hold your breath. You can’t generate your own oxygen just yet.

References:

-       Archibald, J. M. (2005). Jumping Genes and Shrinking Genomes - Probing the Evolution of Eukaryotic Photosynthesis with Genomics. IUBMB Life, 57(8), 539–547. https://doi.org/10.1080/15216540500167732.

-       Chan, C. X. & Bhattacharya, D. (2010) The Origin of Plastids. Nature Education 3(9):84.

-       Johnson, M. D., Tengs, T., Oldach, D. and Stoecker, D. K. (2006), SEQUESTRATION, PERFORMANCE, AND FUNCTIONAL CONTROL OF CRYPTOPHYTE PLASTIDS IN THE CILIATE MYRIONECTA RUBRA (CILIOPHORA)¹. Journal of Phycology, 42: 1235–1246. doi:10.1111/j.1529-8817.2006.00275.x

-       Johnson, M. D., Oldach, D., Delwiche, C. F., & Stoecker, D. K. (2007). Retention of transcriptionally active cryptophyte nuclei by the ciliate Myrionecta rubra. Nature, 445(7126), 426–428. https://doi.org/10.1038/nature05496.

-       Timmis, J. N., Ayliffe, M. A., Huang, C. Y., & Martin, W. (2004). Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes. Nat Rev Genet, 5(2), 123–135. https://doi.org/10.1038/nrg1271.

-       Wisecaver, J. H., & Hackett, J. D. (2010). Transcriptome analysis reveals nuclear-encoded proteins for the maintenance of temporary plastids in the dinoflagellate Dinophysis acuminata. BMC Genomics, 11, 366. http://doi.org/10.1186/1471-2164-11-366.

-       Zhaxybayeva, O., & Doolittle, W. F. (2011). Lateral gene transfer. Current Biology, 21(7), R242–R246. https://doi.org/10.1016/j.cub.2011.01.045.

A Tale of Two Microbes

by Katie Kelly

Growing up amongst vast monocultures of corn and soybean made me appreciate the details. I could easily spend hours outside sitting in the backyard staring at the grass and rocks. The largest rock in my backyard always caught my interest.

Blue-gray circles cover most of the rock (Figure 1). The patches didn’t look to be part of the rock; in fact, they resembled moss, but these patches were not spongy or green. I would pick at these enigmatic patches and cut them up, but they did not possess any apparent roots. They did not produce any leaves or flowers. No stems appeared throughout the year. Over the years, I noticed similar perplexing patches on live trees and decaying wood. My mom told me that this patch was lichen, a living organism. In truth, she was half-right.

Figure 1. The rock of interest. A) Small boulder/large rock from New Lenox, IL. B) Blue-gray patches of interest. Credit: Carrie Kelly.

Lichen is two organisms living together, or “symbiotically.” A lichen species is shorthand for a pair of two microorganisms: an algal species and a fungal species1. The unicellular algal species is referred to as the “photobiont” because it photosynthesizes and provides carbon nutrients to the fungal partner^1,2. Trebouxia species are the most common photobiont in lichens, making up 40% of all observed lichens³.The fungal species is referred to as the “mycobiont” and is responsible for the majority of visible growth of the lichen. Lichen may also contain a third symbiont which is bacterial³.

Lichen diversity is vast due to the huge number of lichenizing fungal species and the ability to have diverse symbiotic partners. Approximately 25% of all fungi are lichenized, with a majority of fungal species members of the family Ascomyceta (members are “ascomycetes”)³. It’s important to note that the estimated number of fungal species on Earth is between 712,000 and 1.5 million^4,5. Even with the most conservative estimate, that’s a lot of lichenizing fungi. Its dominance is evident: lichen is found on every continent, even the barren Antarctica⁴. So no matter where I go, I will find lichen—and photograph them, too (Figure 1, 2, concluding pictures).

Figure 2. Lichen found in Hamilton, MT. Credit: Katie Kelly

This diversity in symbiotic partners leads to diverse morphologies. The visible morphologies of lichens are categorized by their shapes: flat, crust-like lichen are crustose; bead-like clusters are squamulose; leaf-like lichen are foliose; and branched tube lichens are fruticose (Figure 2)⁶. The color of lichen depends on the metabolism of the mycobiont⁷. Whether a lichen is blue or yellow can depend on whether the fungal species is producing sugar chains or amino acids⁷.

Figure 3. Lichen morphology. These panels represent the major morphologies of lichen, respectively: crustose, squamulose, foliose, and fruticose⁶.

Although the visible morphologies of lichens vary between species, the microbiological organization of the photobiont and the mycobiont is largely conserved. Differences in color and morphology aside, the major structure of lichens is fairly consistent across different partners. A cross section of a lichen ‘body’ or thallus shows specific layers: a thin, tightly-packed layer of fungal hyphae; a layer of algal cells able to access light; a large gap with some hyphae, used for gas exchange; and often a portion of fungal tissue which anchors the lichen to its base (Figure 4).

Figure 4. Structure of lichen. The general architecture of lichen is conserved in all of the different morphologies (Figure 3). This consists of the fungal upper cortex, which is what we can see; the algal layer; a more open cavity consisting of mainly fungal hyphae (seen as strings in this figure); and the lower fungal cortex, which binds or anchors the lichen⁸.

The beauty and amazing diversity of lichens masks one of the more sinister traits. Although many believe the photobiont and mycobiont live mutually benefitting each other, the jury is still out. Why? Some researchers claim the fungal counterpart is actually parasitic. The fungal counterpart will wrap itself around the algal cell, penetrating its hyphae into the algal cell wall to take nutrients⁹. Figure 5 demonstrates this invasive ability, the same method plant pathogens use to infect plants⁹. An algal cell can even be crushed by the force of the fungus and die (Figure 5B).

However, the major difference between Figure 5A and 5B is the species of algal cell. Both alga are in the same genus (Trebouxia), but 5A is Trebouxia erici and 5B is Trebouxia gelatinosa. This suggests specificity to relationships between certain species of photobionts and mycobionts.

Although you may be thinking “That poor Trebouxia algal cell!” right now, don’t worry. Some Trebouxia species have been shown to benefit from this association. Research has shown that lichen partners are more able to withstand extreme drying out and oxidative stress than they would on their own². This highly adaptive trait allows them to withstand long periods without water and high amounts of chemical stress. This research suggests how this resilient partnership has flourished on every continent.

My favorite cartoon science teacher always says, “Let’s take a closer look!” and I live by that motto. Lichens are more than meets the eye: a small symbiosis, a mini-battleground for nutrients, a resilient partnership that weathers the storm. It’s amazing what we can see when we look a little closer.

References

• 1. Lisci, M., Monte, M. & Pacini, E. Lichens and higher plants on stone: a review. Int. Biodeterior. Biodegrad. 51, 1–17 (2003).
• 2. Kranner, I. et al. Antioxidants and photoprotection in a lichen as compared with its isolated symbiotic partners. Proc. Natl. Acad. Sci. U. S. A. 102, 3141–3146 (2005).
• 3. Honegger, R. in Fungal Associations (ed. Hock, P. D. B.) 165–188 (Springer Berlin Heidelberg, 2001).
• 4. Hawksworth, D. L. The magnitude of fungal diversity: the 1·5 million species estimate revisited. Mycological Research (2001). Available at: /core/journals/mycological-research/article/the-magnitude-of-fungal-diversity-the-15-million-species-estimate-revisited/EC6F0D9391AA820DE7AEBB043172CB0B. (Accessed: 11th November 2016)
• 5. Schmit, J. P. & Mueller, G. M. An estimate of the lower limit of global fungal diversity. Biodivers. Conserv. 16, 99–111 (2007).
• 6. Morphology of Lichens. Available at: http://www.ucmp.berkeley.edu/fungi/lichens/lichenmm.html. (Accessed: 11th November 2016)
• 7. Nash, T. H. Lichen flora of the greater Sonoran Desert region. (Lichens Unlimited, Arizona State University, 2002).
• 8. Lichens - Basic Morphology. Available at: http://www.arctic.uoguelph.ca/cpl/organisms/plants/terrestrial/lichens/basicmorph.htm. (Accessed: 11th November 2016)
• 9. Ahmadjian, V. & Jacobs, J. B. Relationship between fungus and alga in the lichen Cladonia cristatella Tuck. Nature 289, 169–172 (1981).