Tuesday, December 3, 2019

Deadly but disguised common but concealed small but ill-omened a tale of Leishmania


Before you scroll down:  know that there are pictures of victims of Leishmaniasis in this document. It is important to know what this parasite is capable of but if it makes you throw up your dinner… well then, you’ve been warned. I have placed the more graphic pictures at the very bottom of this document (end of text), so you can still enjoy a good read! These are figures 4, 5, 6 and 7. Viewer discretion is advised.  
            I’ve always been interested in pathogenic organisms. Something about infecting a host much larger than yourself and brining it down to its knees is fascinatingly terrifying. Through my microbiology classes, I’ve had the easiest time memorizing the names of pathogenic organisms and their mode of transmittance and infection. So, when we were assigned to write a short report about a eukaryotic microbe, I knew what category of microbe I was going to look into. Eukarya (plural of eukaryote) are cells that have a nucleus and their name literally translate to true nucleus from Greek. The other major cells type is prokaryotes, those without a nucleus. Eukarya are cells like us (animals), plants, fungi (mold+ mushrooms) and a large protist group; the most famous member of which is the common amoeba. Out of the infectious eukarya, Plasmodium (causative agent of Malaria)was the first organism that came to my mind, but I wanted to use this opportunity to learn about a new organism. At least to me, Plasmodiumand a lot of other famous eukaryotic microbes are a dead horse; I already know so much about them that I can just write a short essay without any research. After about thirty minutes of googling, I chose the organism Leishmania major, though I’ll spend a good chunk of time talking about all Leishmania as well.It is an infectious eukaryote, single celled, it has a cool rather apocalyptical name and it is quite prevalent in our world. Although I suspect I had heard or read about the disease it causes, leishmaniasis, I virtually had no other information about this infectious organism. 
              Evolutionary speaking, we are inclined to naturally fear large predators. Even small, but still potentially dangerous insects can cause a sever reaction in some people. But there is another danger, lurking in an invisible world. Though evolution may not have readied our minds for this world, it did for our bodies. As we speak, there are millions of microbes and viruses desperately trying to invade you and turn you into a microbe-making factory. Can you blame them? Just like the argument humans make when committing an atrocity: “I’m doing it for our children”, they want their progenies to live happily off your body. Sadly for them, your immune system relentlessly destroys almost all of these hardworking microbes. The few that survive either do no harm or are barely scraping by. But then why do we get sick? How can a sickness kill us if our immune system is so good? How some tiny brainless cells can bring a complex organism such as ourselves down? Even with our awesomely equipped immune system? So, sit tight and grab a warm blanket as we dive deep into the cold, remorseless world of one of these pathogens; where no rules except those of Leishmania are abided. 

Figure 1: This figure shows the parasite Leishmania donovani, causative agent of visceral leishmaniasis. Though this the most dangerous kind (look below), most Leishmaniaspecies look very similar. Note the thread like structure at the end of each parasitic cell, which is a flagellum that allows for motility. Picture was taken at 400x magnification using brightfield microscopy. (From here.)
            Leishmania are parasitic eukarya that are present across the globe (fig 1). There are more than 20 species of Leishmania that can infect humans with varying severity depending on species type. The Leishmaniagenus belongs to the order ofTrypanosomatida.This order hosts other human pathogenic species such as Trypanosoma brucei that causes the sleeping sickness and Trypanosoma cruzi that causes the Chagas diseases (2). Like leishmaniasis, both these diseases are transferred by an insect vector to the human host. The severity of infection in other animals varies, though all Trypanosomatida can infect a range of mammals with varying success rates. Frequently, small mammals such as mice can serve as a reservoir for these parasites. The target species here, L. majoris only present in the eastern hemisphere. This species is mostly restricted to Asia, Africa and parts of Europe (1). Regardless of species, humans get infected by the bite of a female sandfly for uptake of a bloodmeal (fig 2 and fig 3). The parasite has a sexual cycle that can only be completed within the female sandfly (fig 2).  Don’t think the female sandfly is very happy to host this unwelcomed guest. The immune system of the female sandfly is also overwhelmed, and its salivary glands are invaded by the parasite (1). However, the sandfly doesn’t get “sick” as it still needs to be able to fly and eat, just to pass the parasite to fresh hosts. We won’t talk about the growth cycle of the parasite, although it is worth noting that it is complicated, and each stage is quite specialized (fig 2). Upon bite of the sandfly, three types of infection are possible: cutaneous, mucocutaneous, and visceral (1). These infection types are restricted by the species type though the demarcations are still blurred as some species seem to cause two or even all three types of infections (1). Now, we will discuss the infection types and the species that can cause them. 
            Cutaneous is the most common type, caused by my target species: L. major. This disease causes serious boils and ulcers to develop on the skin, specifically where the sandfly initially bit the victim. I was surprised to learn that L. majorcan survive uptake by macrophages (3). Macrophages are immune cells in our bodies that eat invading microbes and digest them (phagocytes). In fact, studies have shown the parasite contains several receptors that facilitate its uptake by a macrophage (3). The parasite literally wants to be eaten! Upon ingestion, the parasite survives digestion by the macrophage and turns the tables around: it starts eating the macrophage alive! The parasite will eat and replicate within the macrophage until the macrophage dies. Then, the parasite will lyse the old macrophage and thousands of new progenies will be released into the bloodstream of the host (usually a human). These will all look for fresh hosts: new macrophages that will be infected and the cycle will continue. Luckily, L. majorrestricts itself to the dermis tissue (skin) and does not migrate elsewhere in the body and hence, is not lethal (1). Unfortunately though, the parasite leaves life lasting scars on its host skin which will often entirely deform facial features or other body parts (fig 4 and fig 5). Usually, the victims will face social stigma for the rest of their lives for their disfigured features. 
            The second form, mucocutaneous, is caused by L. tropicaI. As the name suggests, this parasite is predominantly located at tropical regions (1 and 4). Mucocutaneus leishmaniasisfollows a very similar pattern to L. major(cutaneous type) with the exception that mucosal surfaces are also affected along with skin. This is one of the nastiest forms of leishmaniasis (in terms of visible damage done) as the boils and ulcers primarily affect the face (where mucosal surfaces are abundant) and will cause gross disfigurement of lips, mouth, nose and eyes (which may lead to blindness). Additionally, unless controlled quickly, the damage done is often permanent. I have decided against sharing any pictures of mucocutaneous leishmaniasis as they are simply too gruesome for most human beings (myself included). You can look it up online if you desire but you are seriously warned against it! 
            Although the two types of Leishmania I have discussed here so far are nasty, they are not lethal. Though very effective drugs treatments are not available for either the cutaneous or the mucocutaneous form (no cure), our body’s immune system will wipe out the parasite population in about six months to two years. Additionally, the drugs that are currently available can help speed the recovery process and more or less control the damage done by lesions, if administered quickly. However, the third and final form is the most dangerous. This form is one of the most lethal infectious diseases in humans with a mortality rate of near 100% if left untreated. Though malaria takes throne of being the world’s leading eukaryotic parasite killing humans, visceral leishmaniasis comes in second place. This disease is caused by the species Leishmania donovani. In this type, the parasite travels to internal organs such as the spleen, liver and bone marrow (1). Here, the parasite will continue to multiply which will cause massive enlargement of liver and particularly the spleen, which can become even bigger than the liver in some cases (fig 6). If you’ve ever seen a liver and a spleen side by side, the size of the spleen is about 1/3 of a healthy liver. So, patients with this disease often have extremely enlarged abdomens (fig 6). Additionally, the parasite may also actively infect the skin and cause nasty lesions, just like the cutaneous form of leishmaniasis (fig 7). However, the true lethality comes from infection of the bone marrow. The bone marrow is the source of new red blood cells, white blood cells (immune cells) and platelets (blood clotting factors). By lounging at the bone marrow site and quite literally eating the marrow, the parasite severely depletes the body of red blood cells, platelets and most importantly, immune cells (1). The patients will often become anemic, lethargic with sudden bouts of fevers. These symptoms are similar to a malaria infection which may cause a malaria misdiagnosis to be made; which is often a death sentence for the patients. It doesn’t help that the geographical areas that hosts L. donovani often host malaria parasites too, which only increases the chances of a misdiagnosis to be made. However, as mentioned, the immune cells of the body are heavily depleted due to activities of the parasite in the bone marrow. Though it might seem a little unrelated at first, the most famous virus in the world, HIV, shares some similarity with visceral leishmaniasis. HIV kills its victims indirectly by depleting the body of a particularly important immune cell. In HIV infection, it is opportunistic infections that kill the patient not the HIV virus itself. Even the virus of common cold can be deadly if you are HIV positive and not under control. Hence, very much like an HIV infection, in visceral leishmaniasis, the hosts die from opportunistic infections rather than the activities of L. donovani directly. Visceral leishmaniasis is a bit different than HIV infection as unlike the HIV virus, L. donovani can destroys all immune cells and does not focus on one specific type. As you have already understood, visceral leishmaniasis is very deadly. I don’t believe I need to tell you that HIV virus is also deadly and has killed millions of people. It is interesting for me to see that two completely different organisms, one a virus and one a eukaryote, get their super lethality and ability to kill their hosts by targeting the same system: the immunity. Of a particular concern to WHO is a visceral leishmaniosis/HIV super infection. As both these infectious diseases suppress and destroy the immune system, the host is almost guaranteed to die. Sadly, the areas that hosts L. donovani often have prevalent rates of HIV infection too; which only increases the chances of getting the super infection. 
Figure 2: Life cycle of Leishmania species. Note the different names and shapes at each stage of growth. The parasite changes its cellular morphology and gene expression to be more specialized for an infection at each stage. (From here.)
Figure 3: A sand fly biting a human finger. The size comparison allows you to see how small the sandfly is compared to a human. This introduces a particular difficulty in preventing bites as sandflies are small enough to squeeze through most mosquito nets and are difficult to see/feel. (From here.)
            I can’t lie, before I started my research, I didn’t think much of leishmaniasis. Maybe it’s even apparent towards the beginning of this report (while my knowledge was still very incomplete) that I was clearly underestimating Leishmaniaand what it can do. There are lots of pictures depicting the true horrors Leishmania patients undergo, but I must warn you of their graphic nature before you look them up. By being such a nasty, deadly disease, you would except Leishmaniasis to have received abundant funding and research. Sadly, Leishmaniasis is considered a neglected tropical disease and therefore, drugs, treatments and preventive methods are either non-existent or extremely ineffective. Here, I will discuss treatments and prevention for this parasite. 
            To this date, there are no effective vaccines for Leishmaniasis. A couple of vaccines were used in the past but were stopped as the vaccine site itself (usually on the buttocks) would turn into a nasty, slow to heal sore (1 and 2). Hence, the best method is to prevent getting bitten by the sand fly. Recommendation include using insect repellent products, sleeping in closed areas with nets and regular usage of insecticides to kill any insects. Additionally, staying indoors at dawn and dusk may be helpful as sandflies are most active at these hours. As you can clearly see, none of the preventive methods mentioned are truly effective. People that suffer from this disease often live in rural areas with few technological advances. They do not have access to nets, insecticides or insect repellents. Additionally, their houses are not fully enclosed and are easily accessible to insects, especially ones as tiny as the sandfly (fig 3). Staying indoor at dusk/dawn is not an option for these people either as those are the hours that they need to go to work and return back home. The drug amphotericin b deoxycholate, which is actually an antifungal drug has shown to be effective for treatment of visceral leishmaniasis and the two other kind as well. This drug is the only FDA approved drug for treatment of leishmaniasis (4). The drug has to be given everyday for a period of 2-3 months in some cases to prevent relapse of the disease. In visceral leishmaniasis, the drug has to be administered within a period of 30 days to be effective (5 and 6). Unfortunately, the mode of action and the side effects of this drug are poorly understood (1). Renal failures (which often lead to death) have been observed in some patients which may be due to the drug or leishmaniasis (5 and 6). Hence, this drug is far from perfect and requires more research. Additionally, newer drugs are necessary too as some parasite have started gaining resistance to amphotericin b deoxycholate (5 and 6).
            To summarize, why is this parasite so dangerous? What is about leishmaniasis that can prevent our complicated immune system from launching an attack? We have to look into morphology and pathogenicity to answer these questions. There are two main factors that gives Leishmania the ability suppresses human immune system: 1- The ability to move and 2- infecting phagocytotic (macrophages) cells in the body. It is worth noting that Leishmaniacells are all motile by their flagellum (fig 1). Once they lyse their host cells, they can quickly move about the body and infect new cells and tissues. It is this motility that allows them to infect different tissues of the body and move from skin to deep organs like spleen. This motility brings me to my second point, ability to infect immune cells. By moving about quickly, they can evade most immune cells and quickly infect them; it’s more I catch you before you catch me! Most intracellular parasites (the ones that hide within cells) are protected from the immune system to some degree as they are enclosed within the host cell. Our immune system fails to recognize these infected cells until they start encoding wrong cell surface receptors. This parasite has taken this protection to the next level, where it actually hides within the cells that should be responsible for its destruction. The phagocyte, which is usually a macrophage, attempts to do its job and ingest the parasitic cell. This is when its normal mechanisms are hijacked by the parasite and the parasite starts living within the macrophage. Furthermore, the parasite suppresses normal interleukins and other chemicals secreted by the phagocytes. For those who haven’t taken immunology, these interleukins serve as bridge between adaptive and innate immunity. Essentially, they are like little letters (messages) from small guns of immunity (innate immunity) that let the big guns of immunity (adaptive immunity) know it’s time to go nuclear. Our body, like any sensible defense system, uses small guns for small skirmishes and only moves to big ones when it really feels threatened. By blocking the release of these messengers, which would normally be released by a macrophage upon ingestion of a dangerous microbe, the parasite slows down body’s response. That’s the reason it takes such a long time for a strong immune response to be launched and for the parasite to be wiped out (up until a year). As discussed, we saw this slowed response can be deadly in some cases. Another important piece of information is infection of dendritic cells. You can see dendritic cells as postal services of the body. They carry the necessary messages to initiate a massive attack and take it to a place where the big guns of immunity can read it and be activated. Dendritic cells simply chill in the body (at different locations) and ingest different materials (messages) and microbes until they are needed to take certain messages to activate the big guns of immunity. Therefore, by targeting dendritic cells, this parasite essentially paralyzes the postal services of immunity and in turn the adaptive immune system (big guns) for a long time. It’s really genius in a cruel way.  
            So, what have we learned so far? Leishmaniasis is a nasty disease and it kills and disfigures our fellow humans with no regards or mercy. But fear not as WHO and other agencies are slowly working their ways to control and eradicate this disease. As with all of our problems in this world, education is the answer. For many people, they don’t understand how leishmaniasis works, what they can do to avoid it or how to diagnose it in themselves or their animals before things get out of hand. If you want to make a change, there are three options you can take: 1- you can go and study Leishmania for a career. This means you’d have to go to graduate school or medical school and that’s hard and expensive. But if you have the means to do so, it will be worth it as your efforts will lift generations from suffering. 2- Or you can look up leishmaniasis on WHO and see which of their options of preventing this disease fits your interests the best. 3- Which is the last but not the least method: education. Familiarize yourself and those around you with this disease. Don’t scare them by showing them the nasty pictures but do urge them to let a physician have a look at that bug bite which is turning nasty. Remember Leishmaniasis is truly dangerous when diagnosed late. If we can understand it, we can destroy it. Us humans have eradicated and destroyed many obstacles in our history, from travelling in air to smallpox; nothing has stopped us. Leishmania does not stand a chance, I’m sure of it. It will take some time, but we will prevail, we always have and always will. 

Graphic Figures below!

Figure 4: children affected by Leishmaniasis which mostly seems to be cutaneous (the type that affects skin). The sores can usually leave permanent scars and disfiguration which leads to heavy social stigma. (From here.)

Figure 5: child suffering from cutaneous leishmaniasis, the less severe kind affecting only skin. (From here.)
Figure 6: Girl affected by visceral leishmaniasis, caused by L. donovani and the most serious kind. The bloated abdomen area is a sign of inflamed liver and most especially, the super inflamed spleen. (From here.)
Figure 7: Dog affected by Leishmaniasis. The agent here is believed to be the L. donovani (visceral Leishmaniasis). Not only will the animals themselves suffer, but they can also serve as a reservoir for the parasite and eventually pass it on to humans. (From here.)
1.    Ready, P. D. (2014). Epidemiology of visceral leishmaniasis. Clinical epidemiology6, 147.
2.     Jaskowska, Eleanor; Butler, Claire; Preston, Gail; Kelly, Steven (2015): A maximum likelihood protein sequence phylogenetic tree of trypanosomatids.. PLOS Pathogens. Figure. https://doi.org/10.1371/journal.ppat.1004484.g002
3.    Turco, S. J., & Descoteaux, A. (1992). The lipophosphoglycan of Leishmania parasites. Annual review of microbiology46(1), 65-92.
4.    Aronson, N., Herwaldt, B. L., Libman, M., Pearson, R., Lopez-Velez, R., Weina, P., ... & Magill, A. (2016). Diagnosis and treatment of leishmaniasis: clinical practice guidelines by the Infectious Diseases Society of America (IDSA) and the American Society of Tropical Medicine and Hygiene (ASTMH). Clinical infectious diseases63(12), e202-e264.
5.    Thakur, C. P., Singh, R. K., Hassan, S. M., Kumar, R., Narain, S., & Kumar, A. (1999). Amphotericin B deoxycholate treatment of visceral leishmaniasis with newer modes of administration and precautions: a study of 938 cases. Transactions of the Royal Society of Tropical Medicine and Hygiene93(3), 319-323.
6.    Das, A., Karthick, M., Dwivedi, S., Banerjee, I., Mahapatra, T., Srikantiah, S., & Chaudhuri, I. (2016). Epidemiologic correlates of mortality among symptomatic visceral leishmaniasis cases: findings from situation assessment in high endemic foci in India. PLoS neglected tropical diseases10(11), e0005150.

Tuesday, February 12, 2019

Variations on a Theme: Mitosis in Fungi and Animals or, a real cluster-%@!*

by Christopher Molenaar

Greetings, budding biologists! Today, we’re talking about *drum roll* cell division!

I can feel your eyes rolling through the internet. Trust me, though–you didn’t cover this in high school, so bear with me until we get to the fun part. First, we’ll run a crash course on human mitosis, before diving into the odd world of mitosis in single-celled fungi, where cell division can get… weird. Two groups of fungi, ascomycetes and basidiomycetes, have unique ways to divide; not only that, but comparing these organisms to animals suggests an evolutionary history for how mitosis evolved in “us”! Once we describe cell division in human cells, we’ll look at C. albicans(an Ascomycete) and C. neoformans (a Basidiomycete) to see that evolutionary history.

Strap yourselves in.

By this point, you’ve probably taken some sort of high school biology; my guess is you’ve been forced to memorize mitosis (cell division that generates two identical daughter cells) to the point that your mnemonic for IPMAT is ingrained in your mind like any old piece of nonsense trivia. Or, maybe you really enjoyed cell biology, have a strong understanding of cell division, but lack an affinity for social cues and norms. Either way, we’re all very familiar with a figure like this:
We had one cell, and now we have two. Classic mitosis. Notice the nuclear membrane breaking down in Prophase and themicrotubule spindles connecting to the chromosomes. We’ll talk about that later…(Figure from here.)
If you need a brief review, diploid cells (cells with two sets of each chromosome) undergoing mitosis need to 1) condense their genetic material into chromosomes 2) properly separate sister chromatids from these chromosomes into the cellular space of the two new cells and 3) pinch off the cellular membrane to produce two viable daughter cells. There’s a lot more happening, of course, but this is all we’ll focus on here.

In high school biology, I only ever learned about traditional animal mitosis (human mitosis, really); it’s etched in my brain, and yet I never thought to ask any questions about it. Mitosis is this incredibly complex series of interactions between genetic material, the nuclear membrane, the cytoskeleton, and a host of other cell proteins–how did we “get here”? How far back, evolutionarily, does this process go? How much diversity is there for mitosis overall?

In case you were wondering, this is the fun part.

Diagram of kinetochore assembled on
centromere based on electron microscopy.8
To understand the difference between animal and fungal methods of cell division, we need to get into the nitty-gritty of mitosis, focusing on one component of the process: the kinetochore. A kinetochore is a protein structure, built on the chromosome Lego-style from several smaller proteins, that facilitates an interaction between the spindle microtubules and the chromosomes (shown in orange in the image below).2Remember, the spindle microtubules stretch across the cell to bind to the chromosome (shown in green). The kinetochore forms on the centromere (a specific location on the chromosome) as a sort of middle-man for this interaction; in animals, this structure is removed when the cell is not dividing. Generally, the kinetochore has trilaminar architecture: i.e., it has three main layers that interact with different components of the mitotic process.2The inner layer, for example, interacts directly with the chromosomal DNA, and outer proteins interact with microtubules.2  This structuring is mostly conserved in eukaryotes (plants, fungi, animals, etc.), meaning that it’s common across these organisms.3Yet the timingvaries greatly, as do surrounding steps in the mitotic process.

Cell division in C.
looks a lot like
division in S. cerevisiae,
shown here.2
What do I mean by “surrounding steps”? While the kinetochore is being assembled and the microtubules are extending, the nuclear membrane (or  envelope) needs to undergo some alterations for effective mitosis–in humans, at least. As shown in the image above, the nuclear membrane breaks down during mitosis in almost all animals–this is termed “open mitosis”.3,4However, this isn’t the only possibility! Other eukaryotes, like fungi, undergo closed mitosis, where the nuclear membrane never breaks down.3,4

Take Candida albicans, for example. C. albicans is a fungi in the Ascomycete phyla, a group that contains other budding yeasts like Saccharomyces cerevisiae (the yeast used for beer, wine, bread, and a bunch of other foods). When diploid C. albicans cells divide by mitosis, the nuclear membrane stays completely intact throughout the process, stretching into the daughter cell space before pinching into two nuclei (shown right)5. Additionally, the kinetochore is fully assembled and attached to microtubules for the entire cell cycle–even when cells aren’t dividing!2Pretty weird, right? When these organisms divide, the separation of genetic material into daughter cells relies on microtubules withinthe nucleus, rather than microtubules coming from outside the nucleus (as in animal mitosis). This is an example from the Ascomycetes; what about other fungal phyla, though?

Well, that’s what got me interested in this topic. A fungus that I study, Cryptococcus neoformans, follows some pretty unique rules compared to the Ascomycetes I just mentioned. C. neoformansis a Basidiomycete–this phylum, sometimes called the club fungi, includes the typical mushroom you would put on a salad. C. neoformans, though, is another single-celled organism, like C. albicans. This fungus has a lot of interesting behaviors, but before I go off on a tangent, let’s talk about how it divides and assembles the kinetochore structure.

Remember, in humans and almost all animals, the nuclear membrane is completely degraded to allow the chromosomes to migrate to opposing sides of the dividing cell; the kinetochore is assembled during mitosis, and is removed in non-dividing cells.2,4However, in some ascomycetous fungi, the nuclear membrane never breaks down, and instead gets pinched off like the cellular membrane during telophase. Additionally, the kinetochore can remain on the centromere throughout the cell cycle.2,6But what does C. neoformans do? Well, it’s a little of both.

In C. neoformans, kinetochores assemble just before mitosis, like in animals.5The inner kinetochore proteins remain on the centromere throughout the cell cycle, but the middle and outer proteins only assemble when the chromosomes cluster during mitosis.2Not only do these structures have different timing of assembly compared to ascomycetes–they take up a completely different location! In Ascomycetes, the centromeres of different chromosomes are clustered in a single location throughout the cell cycle (until chromosomes are split into two recipient daughter cells); as mentioned, the microtubule-kinetochore-centromere complex is maintained even when cells are not dividing in most of these fungi.6,7However, in Basidiomyceteslike C. neoformans, the centromeres are not clustered; instead, these regions of the chromosome are spaced out around the periphery of the nucleus, more similar to the arrangement of genetic material in some animals.2
This is C. neoformans undergoing mitosis. Three things are shown here: degradation of the nuclear membrane (red), clustering/declustering of the kinetochores and centromeres, and microtubules originating from outside the nucleus to direct the genetic material.2
Lastly, a major difference between basidiomycete and ascomycete mitosis, and a similarity between the former and animal mitosis: theC. neoformansnuclear membrane is partially degraded during mitosis.2As the microtubule spindle migrates to the daughter cell (shown at right, next to the star), the nuclear membrane is broken to allow this migration. This is a significant variation from conventional closed mitosis in fungi, where the membrane stays completely intact throughout the entire cell cycle.

In a recent study of C. neoformans, it was suggested that this variety of mitosis is highly reminiscent of animal mitosis, and that mitotic events associated with animals (like kinetochore assembly, opening of the nuclear membrane, and clustering/declustering of centromeres) evolved in the fungal kingdom.2These distinctions in mitosis between ascomycete S. cerevisiae, basidiomycete C. neoformans, and humans are laid out nicely in the image below.
Comparing the mitotic process of an ascomycete, the
basidiomycete C. neoformans, and human cells.2
So there it is: some added spice to traditional human cell division! Mitosis isn’t just an acronym that you have to remember–it’s a complex process with a lot of diversity within eukaryotes! Unfortunately, a lot of foundational biological concepts get reduced and packaged so that they’re more manageable for testing, and cell division is certainly one of these. In biology, though, the more you learn about a concept, the more fascinating it is! Mitosis can be achieved via a spectrum of approaches; understanding the connections between these can give a lot of insight into evolutionary relationships. Perhaps next time I see a cell division figure, I’ll give less groan and more glory to a genuinely incredible biological process.


2.  Kozubowski L, Yadav V, Chatterjee G, et al. Ordered kinetochore assembly in the human pathogenic basidiomycetous yeast Cryptoccous neoformansmBio2013, 4(5) 1-8. 
3.  Meraldi P, McAinsh A, et al. Phylogenetic and structural analysis of centromeric DNA and kinetochore proteins. Genome Biology2006, 7(23). 
4.  Przewloka M, Zhang W, et al. Molecular analysis of core kinetochore composition and assembly in Drosophila melanogasterPlosOne2007. 
5.  Kozubowski L, Heitman J. Profiling a killer, the development of Cryptococcus neoformansFEMS Microbiology Reviews2012, 36(1), 78-94. 
6.  Thakur J, Sanyal K. The essentiality of the fungus-specific Dam1 complex is correlated with a One-Kinetochore-One-Microtubule interaction present throughout the cell cycle, independent of the nature of a centromere. Eukaryotic Cell2011, 10(10), 1295-1305. 
7.  Jin Q, Fuchs J, Loidl J. Centromere clustering is a major determinant of yeast interphase nuclear organization. Journal of Cell Science2000, 113, 1903-1912. 
8.  McEwen B, Dong Y, VandenBeldt K. Using electron microscopy to understand functional mechanisms of chromosome alignment on the mitotic spindle. Methods in Cell Biology2007, 79, 259-293.

Monday, December 10, 2018

Priming the Gut: How Organisms Inside Us Can Fight Our Battles

by Nicholas James Walsh

             Microorganisms live almost everywhere imaginable, from the bottom of the ocean, to outer space, to deep inside our bodies. They even play roles in protecting ourselves, inside and out. Microbes live on our skin, hair, mouths, and between our toes, and these miniature creatures often staves off other dangerous microbes that could potentially infect us. This competition puts these “good microbes” on our side, defending us like a small army, who get the benefit of our nutritious micro-environments. Even in the seeming inhospitable environment that is our gut, these microbes, from yeast to bacteria, can flourish and compose what is deemed the “microbiome.” Controlling everything from our weight to our mental health (1), these microorganisms play a larger role in the daily functioning of human beings than previously believed. Imbalances in the usually robust gut microbiome can result in fluctuations in body fat percentage and even alter the signals coursing through our brains.
             It is no surprise, then, that new data constantly emphasizes the importance of retaining a healthy, balanced gut microbiome. One of the main players in the well-being of the gut environment is the microbe Cryptosporidium parvum. C. parvum is a eukaryote, which means it is arguably more similar to human cells compared to the other bacteria in the gut. However, certain infections with C. parvumcan result in an enteric disease called cryptosporidiosis, which causes small intestine disturbance and watery diarrhea. The infection process is common in malnourished individuals, since low-protein diets can lead to a decreased immune response to certain infections—notably cryptosporidiosis (2). It is these populations that tend to have incomplete diets who are most likely to experience dangerous infection with C. parvum. 
             This may seem paradoxical, because the same organism that is vital in maintaining the well-being of the gut can also cause a potentially fatal infection. This phenomenon is seen in many different organisms in the gut, notably in the notoriousEscherichia coli, or E. coli. Various circumstances can lead certain microbes to become infectious, as many strains of these organisms can produce different levels of beneficence or toxicity. For instance, E. coli is abundant in the gut, but a certain strain that has a unique antigen, or small protein, causes massive food poisoning, even to the point of death. This “O antigen” strain is very similar to healthy E. coli, but are different in this small, extracellular protein, which causes the human body to react very negatively to the bacterium when it proliferates in the small intestine (3).
             How then, do we use this to our advantage? Studies have shown that “priming”, or preparing, the gut with helpful bacteria or protozoa can stave off certain common gut infections. A novel technique, called the fecal microbiota transplant, can transfer healthy microorganisms into our guts, where they will take hold and produce a desired result. This can be conducted through both oral and anal methods, which have shown to be approximately equal in efficacy. 
             A famous study showed that when skinny mice were given high-dosage antibiotics (to clear the gut of bacteria), and quickly administered a fecal microbiota transplant of “obese mice” gut flora, the skinny mice develop mild-to-severe obesity, as seen in the figure below (4). From these studies, and others like it, we have gained an understanding of the processes that are heavily controlled by the microorganisms that live inside us. Not everything is determined purely through genetic luck of the draw, but our environment, including the small beings that live within us, can have strong impacts on our physical and mental well-being.
Figure 1. The scheme above shows the process by which a fecal transplant
 can affect the body fat percentage of mice. The microbiome of obese mice can
cause lean mice to accumulate large amount of fat when their microbiomes
 are placed in a sterile lean mouse microbiome.
             In the case of cryptosporidiosis, low-dosage exposure to C. parvumto the human gut is showing promise as a new method to alleviate Cryptosporidium-specific diarrhea in patients, particularly affected children in generally poorer populations.
             To conduct these pseudo-surgeries, the human gut is “blasted” with antibiotics. This kills off a strong portion of the microbiome, which creates gut vacancy that can quickly be occupied by the first organism that can find a niche in it. Though this may seem like a dangerous process, considering the aforementioned importance of maintaining a healthy microbiome in the gut, studies have shown that the composition of the gut is generally very robust, even in responses to stressors like antibiotic hits and diets containing high quantities of sulfur. After disturbance, the makeup of the gut is elastic, and “whips” back to its normal composition in a matter of days, like a slow rubber band. These transplants work by taking advantage of the time window available during this period, where niches in the gut are free, and instead of reverting back to the original gut composition, it becomes slightly modified (5).
             Even in the last ten years, new fecal microbiota transplant techniques have alleviated diseases such as Clostridium difficile infection and diabetes. Though it seems strange, these fecal transplants involve using one of the most unlikely substances to improve your heath—poop. In fact, thousands of labs arounds the United States are using fecal samples to improve the health of individuals. A fecal transplant is similar to a liver or lung transplant: a “defective” gut microbiome is fully or partially replaced with the gut microbiome of a healthy, non-infected individual. Often a general fecal sample is taken from a young, healthy individual with a balanced diet and fully replaces the gut microbiome of the patient, as seen in the image below. This is the method being used to treat C. difficile infection and the like. It is the standard, less risky method to cure generalized gut infection, yet not all enteric diseases can be tackled in the same exact simple manner.
Figure 2. The general process by which a fecal microbiota transplant (FMT) occurs.
Simply, the microorganisms in the gut of healthy individuals is placed in a
sterile gut of a patient with a gut-affecting condition.
             The case of C. parvum infection is slightly different—its treatment involves giving low-dosage C. parvum gut injections, which seems counterintuitive. However, the logic applies here is similar to that of your garden variety flu shot. Many shots involve injecting a harmless/modified, or dead, version of the virus or bacterial toxin into the human bloodstream, and the immune system goes through a process of developing memory immune cells to tackle a future infection that mimics that of the flu shot. Because the infection does not pose a biological threat, the immune system can take care of it efficiently and prevent future infection (6). 
The microbiome priming technique follows a similar line of logic. Most infections occur in those with low-protein diets, which leads to a decrease in certain immune responses. Here, these responses are called Th-1 responses and cytokine responses. These interactions by the immune system are critical in halting C. parvum infection. Restoring these responses is key in stopping lethal cryptosporidiosis (6).
            With this in mind, two routes appear feasible in ridding infection: the first is reestablishing a protein-heavy diet, and the second is priming via C. parvuminjection. Many neglected or lower socio-economic populations are unable to maintain a balanced diet, and many lifestyle choice involve low-protein intake, as well. This leads many groups of people to be susceptible to potentially fatal protozoan infection by C. parvum. Often these diets changes are not possible or are otherwise rejected. Therefore, a pragmatic approach to reducing the incidence of cryptosporidiosis is fecal transplantation in these populations. So, use of fecal microbiota transplantation can be a plausible option for those who cannot alter their diets to fit a protein-heavy intake. 
             Not only are these findings about C. parvum infection clinically applicable, but it may pave a new common route for treating gut infections that are tied to microbiota imbalances. Traditional methods to improving gut health usually involve dieting techniques, medication, or other lifestyle changes. These prescriptions may be effective in cases, but disturbances in the gut microbiome are extremely difficult to fix due to their elasticity. With the case of dieting, studies in the past have shown that most diet changes do not permanently alter the gut composition. Even high dairy or sulfur diets over the course of weeks hardly change the organisms abundant in the gut, and when they do change, it seems to be temporary. Exercise and other anti-inflammatory lifestyle approaches are also common route in treating gut infection, particularly related to colitis. Yet these methods are not necessarily a permanent or even effective way to ameliorate gut disturbance symptoms. New evidence related to cryptosporidiosis infection is showing that these “gut vaccines” may be a route for reducing gut disease incidence. Most vaccines are typically seen as bloodstream injections involving dead or broken viruses, but gut priming appears to follow a similar logic and could be just as valid of a medical treatment.
             Because the impact that the gut microbiome has on the human body—which is a growing library of knowledge recently—is seems to be possible to manipulate the makeup of the gut to improve many conditions. Here, it is intriguing that priming the intestines with C. parvum leads to alleviation of the lethal infection caused by the same protozoan. This could pave the way to treating other gut infections related to common microbiome species, like E. colior C. difficile, which appear to be more commonplace in the wake of an aging, susceptible population (figure 3).
Figure 3. The rate by which individuals in the USA are affected with gut-related diseases appears to be increasing with time, particularly in older populations. Because many treatments to other diseases, like cancer, involve unintentional gut dysbiosis, colitis rate seem to increase, which makes FMTs a popular and effective solution to gut dysbiosis.

Works Cited:

1)     Mangiola F, Ianiro G, Franceschi F, Fagiuoli S, Gasbarrini G, Gasbarrini A. Gut microbiota in autism and mood disorders. World J Gastroenterol 2016; 22(1): 361-368
2)     Cryptosporidium Pathogenicity and Virulence; Maha Bouzid, Paul R. Hunter, Rachel M. Chalmers, Kevin M. Tyler; Clinical Microbiology Reviews Jan 2013, 26 (1) 115-134; DOI: 10.1128/CMR.00076-12
3)    Sarkar S, Ulett GC, Totsika M, Phan M-D, Schembri MA (2014) Role of Capsule and O Antigen in the Virulence of Uropathogenic Escherichia coli. PLoS ONE 9(4): e94786. doi:10.1371/journal.pone.00947861. 
4)    Kulecka M, Paziewska A, Zeber-Lubecka N, Ambrozkiewicz F, Kopczynski M, Kuklinska U, Pysniak K, Gajewska M, Mikula M, Ostrowski J. Prolonged transfer of feces from the lean mice modulates gut microbiota in obese mice. 2016;13(1):57.
5)     Kelly CR, Khoruts A, Staley C, Sadowsky MJ, Abd M, Alani M, et al. Effect of Fecal Microbiota Transplantation on Recurrence in Multiply Recurrent Clostridium difficile Infection: A Randomized Trial. Ann Intern Med. ;165:609–616. doi: 10.7326/M16-0271
6)   Bartelt LA, Bolick DT, Kolling GL, Roche JK, Zaenker EI, Lara AM, et al. (2016) Cryptosporidium Priming Is More Effective than Vaccine for Protection against Cryptosporidiosis in a Murine Protein Malnutrition Model. PLoS Negl Trop Dis 10(7): e0004820. doi:10.1371/journal.pntd.0004820