Naegleria fowleri: Thanksgiving in Your Brain

Picture this scenario.  It is a hot summer day and you are hanging out with your friends at the local Walmart causing nothing but trouble trying on clothes you are not going to buy, cruising around on the handicap carts, and playing freeze tag.  Being such a sweltering day you decide to give the Walmart employees a break by heading over to the lake to cool off and take a dip.  Arriving at the beach you shut the car off, swing open the door, and race to the water.  Showing off for your friends you take a giant leap at the edge of the water, go airborne, super-man, and then finish by gracefully diving into the water.  Unfortunately for you, you were not graceful enough and receive a nice sinus flushing.  Other than a minor lingering headache that hangs around the rest of the day, you are fine, for now.  Normally they say what you do not know does not hurt you, but in this case it will kill you.  Funny how a day full of fun and pranks can turn in to one of unbearable pain, vomiting, and death, all thanks to the water-borne amoeba Naegleria fowleri.

N. fowleri life stages

            Unbeknownst to you, the water that flushed your sinuses contained the free-living amoeba N. fowleri.  To make matters worse, it has begun burrowing its way through your skull towards your brain, where it will feast.  N. fowleri was first cultured and documented by the medical scientists M. Fowler and R. F. Carter in Southern Australia 1965 (1).  They, as is common practice today, extracted the amoeba from the dead body of a N. fowleri victim.  The problem with this practice is the patient is all ready dead, so they cannot be helped.  Interestingly N. fowleri exists in three diverse morphological states: the classic amoebic trophozoite, the flagellate, and the cyst.  Trophozoites are the actively breeding, feeding, and infecting morphology of N. fowleri.  In this form it is motile via active sinusoidal/limacine (oscillating) locomotion and reproduces strictly by binary fission (2).  Trophozoites transform into the flagellate form when ionic concentrations are changed, but this has only been observed under laboratory conditions.  Common in nature however is the highly resilient cyst form of N. fowleri, which Trophozoites transform to under nutrient starvation or harsh conditions (1).  Understanding the variant morphological forms and how they lead to a N. fowleri infection may help shed light on possible treatments.

Food-cup shown by red arrow (1).    

            Upon arriving in your nose, and if conditions are right, the amoeba will find itself at the olfactory neuroepithelium, the thin tissue loaded with nerve endings that connects your nasal passageway to your brain.  At this point it is believed that your own cells phagocytose, or engulf, N. fowleri cells, bringing them inside your own cells possibly to generate an immune response (2).  Depending upon the virulence of the N. fowleri strain inoculated in your nose, the amoeba will begin burrowing its way towards your brain, evading an immune response.  It literally follows the nerves directly to the brain, eating tissue as it goes.  In culture N. fowleri has indistinguishable surface structures called “food-cups” that are believed to act as a mouth for the organism, nibbling away at tissue and engulfing it piece by piece in a process called trogocytosis (3).  Within a few days of burrowing N. fowleri will reach your brain, upon which it will kill you.  Reaching the brain is like thanksgiving for the amoeba, as it literally goes on a feeding frenzy gorging on your neural flesh.  N. fowleri also begins reproducing at this point, increasing the number of trophozoites in your skull, amplifying the effects of the infection.

            Upon reaching the cranial cavity and reproducing the infection is now considered primary amebic meningoencephalitis (PAM).  PAM is described as a rapidly fatal disease of the central nervous system (1).  The problem with a N. fowleri infection, PAM, and for you is that detecting the infection early enough is difficult as the symptoms associated with PAM are extremely vague.  With PAM you may experience an intense headache, fever, nausea, vomiting, stiff neck, and potentially a few seizures, all of which would not lead you to the idea that a microbe is slowly burrowing into your brain killing you.  Adding to the confusing symptoms is the fact that you are a healthy, immune-competent young adult; this distinguishes N. fowleri from other free-living amoeba in that it is not an opportunistic infector, but a direct pathogen (2).  In the end you will die a brutally painful death as your brain hemorrhages and you slowly bleed to death

            Depending upon the virulence of the N. fowleri strain eating its way into your brain, your body may or may not develop an effective immune response on its own.  Your first defense is going to be the complement system.  If you are lucky complement will bind to N. fowleri cells, opsonize it (cover the cell surface), and potentially destroy it.  If you are not lucky N. fowleri will simply absorb the complement proteins, negating its immunological effect, or it will express regulatory surface markers that avoid the attention of complement all together.  Next in line are your phagocytic leukocytes, macrophages and neutrophils.  Depending upon the virulence of the amoeba your leukocytes will either destroy the N. fowleri, or be destroyed.  Your body does generate plenty of antibodies against N. fowleri, but without competent cells to react to the antibodies effects they are useless (1).  If your body fails to eliminate the N. fowleri infection on its own you are out of luck.  Currently there are no clinically used drugs that affectively treat a N. fowleri infection.  Doctors have tried using various combinations of Amphotericin B and fluconazole, potent anti-fungal drugs that both target the membrane bound molecule ergosterol, along with rifampin, a drug that prohibits DNA transcription.  The combination has shown promise in the laboratory, but has seen little effect in patients (1).

            While N. fowleri is found in just about every warm body of water possible, the frequency of infection is relatively low with about one case per year (2).  The only way to avoid a possible infection is to stay out of contaminated water all together.  While that is not entirely feasible for some people, I would say you can play the odds, enjoy the water and pray that you are not the one person a year who is brutally killed by the brain eating amoeba, Naegleria fowleri.

Submitted by KH

References:

1.              Cabral, Guy A. "The Immune Response to Naegleria fowleri Amebae and Pathogenesis of Infection." FEMS Immunology & Medical Microbiology. 51.2 (2007): 243-259.

2.              Visvesvara, Govinda S. "Pathogenic and Opportunistic Free-living Amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea." FEMS Immunology & Medical Microbiology. 50.1 (2007): 1-26.

3.              Marciano-Cabral F.  “Cytopathology of pathogenic and nonpathogenic Naegleria species for cultured rat neuroblastoma cells.”  Applied Environmental Microbiology.  51. (1986): 1133-1137.

Euglena: A Paradoxical Eukaryotic Species Serving as a Unique Tool in Research

The complex and unique nature of Euglena makes it a useful experimental tool for research. It is unique because it is a protist that shares both animal-like and plant-like characteristics that scientists make use of to study the evolutionary aspects and biological behaviors of both plants and animals. Many experimental studies benefit from the animal-like behavior of Euglena in the dark and its plant-like behavior in the light. In the light, Euglena is actively photosynthetic and green. When conditions are altered to become dark, Euglena is rendered colorless. However, re-exposure to light will slowly cause Euglena to revert back to its green color resuming its photosynthetic activity. The experiments geared towards studying the molecular basis for these changes could be used to understand the processes of “photosynthesis, phototropism, vision and communication” due to the cellular and structural features present in Euglena (1).

When observing euglena through a microscope, one could expect to see a splendidly green spindle-shaped unicellular organism exhibiting peculiar wormlike contractions, popularly known as ‘euglenoid movements’. Euglena moves due to the presence of a flagellum that projects from its anterior end and runs along the side of its body. This structure is made up microtubules that enable euglena to move forward and rotate, very often following a corkscrew path. The green color is caused by the presence of chlorophyll containing chloroplasts, which are photosynthetic plastids dispersed throughout the cytoplasm. However, there are many variants of euglena that are also colorless, red, yellow or brown (1).

Another fascinating structural aspect of euglena biology is the location and function of a photoreceptive organ called the eyespot or stigma that appears as an orange-red region near the flagellum. This orange-red stigma is what gives this species its name Euglena, which literally translates to true eyeball. The flagellum and the eyespot act together as a singular unit in the detection and movement towards light for the photosynthetic activity of this organism, thereby helping it synthesize carbohydrates from carbon dioxide and water, an autotrophic mode of nutrition, like any green plant (1).

However, euglena also has the capacity for heterotrophic means of obtaining its food, similar to animal-like protists known as peranema, by engulfing particles of food found in its habitat such as still pools and ponds, where they often give a greenish color to the water. As well as being able to move and feed like animals, Euglena also lack a cell wall, a very well-known characteristic of plants. Instead their bodies are surrounded by a flexible structure called a pellicle.

Euglena gracilis and Euglena viridis are commonly studied species of Euglena. However, over thousand species of Euglena have been identified. Euglena can yield useful research information as it is able to adapt to variable environmental conditions such as changes in the temperature, chemical content in its environment and light or dark conditions (1).

It serves as a model organism for the study of the use of light by living systems because molecular studies utilizing its chloroplasts are feasible. Its reaction to light makes it a highly sensory cell and helps answers questions about the relationship between the receptor (the eyespot) and the effector (its flagellum).The mechanism of the eyespot and flagellum is analogous to the reflex action. Experiments involving the eyespot have revealed that the energy absorbed by Euglena is directly proportional to its mobility (1).

Other studies have shown that the pigment present in the eyespot of Euglena gracilis has similarities to rhodopsin (2). Consequently, deeper analysis of the eyespot pigments compared to the visual pigments in the retinas of animal cells may help shed light on the photoreceptor systems of animals. Similarities between the phototactic behavior of Euglena and the visual process in animals may also exist (1).

Euglena in research also has the potential to provide significant evolutionary information about introns of chloroplasts and transitions from plant like characteristics to animal like characteristics.

Introns are non-coding regions of DNA. The evolution of chloroplast introns and twintrons (occurrence of introns within introns) gives valid genetic information about the intron evolution theory. The phenomenon of twintrons may have occurred later in evolution by the insertion of one or more introns into existing introns. The chloroplasts for Euglena gracilis has been identified as the richest source of introns and are used to study the proliferation of group II and group III introns. By looking back in history about specific introns and twintrons using the Euglena plastid lineage it is possible to find out if introns are ancestral or derived traits (3).

Different theories of evolution have been put forward regarding plants and animals. One popular theory believes that an early stem organism with the ability to photosynthesize could have been the precursor of plants and animals. Its ability to photosynthesize would have given it the advantage to derive nourishment in conditions of organic food scarcity. After, the establishment of plant life, when organic foods became plentiful the same organism may have transformed to incorporate animal like characteristics similar to those seen in euglena under dark conditions by loss of its chlorophyll. A different theory suggests that Euglena is representative of a group of organisms containing both colorless and colored forms, from which plants, fungi and animals evolved separately. The evolution of plants may indicate a chance encounter progressing into a symbiosis (1).

Regardless of whether Euglena is closer to animals or plants, the possession of characteristics belonging to both plants and animals show its potential in research as a useful organism giving information about chloroplasts in photosynthesis, pigment synthesis, visual process in animals and its cellular contents pertaining to growth and functional physiology (1). Euglena studies shows that at a molecular level, animals and plants share a lot of similarity forming a common basis for living processes. However, it also emphasizes to all scientists that the answers to the evolutionary process do not easily come by and requires detailed analysis and use of unique organisms like Euglena as research tools.

References

1. Jerome J. Wolken, Euglena. An Experimental Organism for Biochemical and Biophysical Studies. Rutgers (New Jersey) 1961. Rutgers University Press.

2. James T.W., Crescitelli F., Loew E.R., McFarland W.N. The eyespot of euglena gracilis: a microspectrophotometric study. Vision Research.1992; 32: 1583-1591.

3. Thompson MD, Copertino DW, Thompson E, Favreau MR, Hallick RB. Evidence for the late origin of introns in chloroplast genes from an evolutionary analysis of the genus Euglena. Nucleic Acids Res. 1995; 23: 4745–4752.

Move Aside Arsenic Bacteria

By Amanda Ruben

                  What is the most indestructible thing on Earth? Some things may come to mind such as nanotubes, diamonds or Rocky Balboa’s jaw. All three of these are good guesses, but if you talk to any biologist the answer would be a water bear. These water bears are also known as tardigrades. Now you may be wondering what conditions are so horrendous or harsh that biologists would put this organism on a pedestal. Space vacuum, solar radiation and extreme temperatures are a few conditions that come to mind, but first where can we find these organisms on Earth and what are they closely related to?
                  Tardigrades are microscopic invertebrates with a well-developed organization. They have a brain, muscles, reproductive organs, osmoregulatory organs and sensory organs. Tardigrades inhabit a variety of environments found worldwide. These habitats can range from aquatic to terrestrial to limno-terrestrial which is an environment that frequently dries out.
                  Tardigrades are most closely related to Arthropoda and Nematoda. There are three classes of Tardigrada: Heterotardigrada, Eutardigrada and the controversial Mesotardigrada. The Mesotardigrada is controversial as it only contains a single species which was isolated from a hot spring in Japan. However, the hot spring or specimens no longer exist due to an earthquake disruption. Tardigrades are thought to have evolved within a marine environment, and the various mechanisms behind adaptive tolerances these organisms withstand have yet to be investigated.
                  As stated above, some of the extreme environmental stresses include a space vacuum, solar radiation and extreme temperatures. These are just a few of the conditions in which tardigrades can survive; nonetheless a combination of these stresses cannot defeat a tardigrade. Previous experiments have shown the amazing survival rates of these organisms and their offspring. A space vacuum is similar to desiccation. One study determined a space vacuum had no significant effect on the egg-laying or hatching as compared to the control organisms that did not undergo desiccation. In order to survive desiccation, the organism closes into a ‘tun’. A tun is when the organism retracts its legs and contracts longitudinally into a ball. During the tun phase, metabolism is at nearly a stand still allowing the organism to survive. Once hydration occurs, the organism expands and extends its legs, and metabolism is restored. Tardigrades have been shown to survive for up to 10 years in anhydrobiosis form, which is extreme dessication and this has been shown to have no effect on the production of viable offspring.

This is an image of a water bear forming a ‘tun’ on the right
side in which extreme dessication results in an ametabolic state.    

                  Solar radiation is also an extreme environmental stress that has not been able to knockout all tardigrades. Some species have lowered survival and fitness, but others are able to produce viable offspring even with a combined treatment of radiation and space vacuum.  Another profound question of radiation and space vacuum exposure illustrates how the configuration of DNA survives.  Experimentation of these conditions may help understand changes in DNA configuration and repair which can be applied to various diseases to humans.

                  Extreme temperature is another environmental stress which is no match for the tardigrades.  They have been proven to withstand temperatures of extreme heat and cold.  For example, tardigrades withstand 151°C and can withstand 1 degree above absolute zero for a few minutes.  The mechanisms behind this rapid adaptation have yet to be understood, but may be very insightful for understanding how these organisms can withstand such temperature fluxes.

                  Tardigrades now belong to an elite group of organisms which had the opportunity to become an astronaut, endure the elements of space and live to tell the tale.  As mechanisms behind these environmental adaptations are further studied, the knowledge gained could have many benefits to the human race and our own health problems and diseases.  Therefore, move aside arsenic bacteria.

References

(1). Jonsson, K. et.al. (2008). Tardigrade survive exposure to space in low Earth orbit. Current Biology                   18 (17): R729-R731.

(2).  Bertoliani, R. (2001). Evolution of the reproductive mechanisms in tardigrades-A Review. Zool. Anz. 240: 247-252.

(3). Mobjerg, N. et. al. (2011). Survival in extreme environments-on the current knowledge of                   adaptations in tardigrades. Acta. Physiol. 202: 409-420.

Blastomyces dermatitidis: A review of a deadly disease from a small spore

Amongst the pristine beauty of the northern end of the Mississippi and the Great Lakes lies a deadly yeast that is responsible for infecting more dogs in the Midwest than any other fungus. Blastomyces dermatitidis is the causative agent of the disease Blastomycosis, which can infect lungs, eyes, lymph nodes, bone, CNS, and skin. Even after administration of an antifungal, mortality rate is conservatively 40% (Brömel et al 2005). The clinical signs of this infection are common symptoms to most diseases. Dogs have a fever, they lose weight, stop eating and have low energy, and therefore many owners do not think to get their pet immediately treated. If the fungus is not treated within its early stages, a more rigorous and expensive treatment must be performed for close to a half of a year. In addition to the shear amount of medication needed to rid the animal of the fungus, the antifungal itraconizole costs $3,717, which is three times more then the antifungal used to treat other less serious infections (Mazepa 2011). Because of the lack of insurance, cost can determine whether or not an animal is treated. Without medication, a dog will undoubting succumb to the infection.

Blastomyces dermatitidis exist as a branching fungal hyphae when found in nature. It typically resides in rotting wood, mud, sand and animal wastes near a body of water (Brömel et al 2005). Many infections are diagnosed shortly after the owners take trips with their dog up north to camp or hunt. Infection occurs when a dog inhales spores produced by the fungi’s sexual cycle, so therefore it’s not surprising that the dogs that are infected are typical hunting dogs; a large, intact, male dogs. The germination of the spore, known as conidita, is caused by the rapid change in temperature when entering the body of a dog (Brömel et al 2005). Within the dog, the newly formed yeast triggers an immunological and inflammatory response in which the phagocytes and complement proteins bind B. dermatitidis in attempts to destroy it. Interestingly, B. dermatididis show better growth within the macrophages and other phagocytes then outside the cell. Giles et al. discovered that this enhanced growth was aided by the release of a canine soluble factor, which increases adherence to the walls of the macrophages. Although the factor that enhances cell growth is unknown, the adherence of the yeast to the macrophages protects B. dermatitidis from being degraded by the phagocytes. In addition to the resistance of host macrophages, B. dermatitidis produces melanin. This virulence factor makes the yeast less susceptible to antifungals such as amphotericin and fluconazole. Fortunately, itraconizole is not affected by the presence of melanin (Mazepa 2011).

Early diagnosis of Blastomycosis is pivotal due to the fungus’ ability to become systemic quickly, which can drastically affect the chance of survival.  Because the yeast is carried within the host’s macrophages, the immune system spreads B. dermatitidis and infects other areas of the body such as the skin and eyes. Serosanguineous (blood and pus) drainage can occur from the lesions, which appear on the nose, face, back and nail bed of the animal (Greene 2006). When Blastomycosis infects the eyes, excess of blood vessels, constriction of the eye, and watery swollen cornea are clinical signs. Severe infections can cause the lens of the eye to rupture, consequently resulting in the removal of the eye (Brömel et al 2005).

The two most severe forms of Blastomycosis are infection of the lungs and the central nervous system. Blastomycosis is most commonly seen within the lungs because it is the initial site of germination. Severe pulmonary infections result in a 50% mortality rate within the first seven days of treatment. If the infection spreads to the CNS, very few survive (Greene 2006). An acute pulmonary infection can be seen in an X-ray in which the lungs look cloudy and opaque from the plaques of yeast living amongst the cells.  But to truly know if the patient has Blastomycosis and not a less serious infection, the veterinarian has to see the yeast itself.

The different strategies in diagnosing Blastomycosis depend on site of infection. Tracheal washes are a common procedure to obtain an appropriate sample within animals with a pulmonary infection. The protocol of a wash consists of syringe placed down the trachea and sterile saline coats the tissue surface. The saline then is quickly collected with the syringe and can be analyzed for budding yeast. Recently, a urine test has been a tool to be able to diagnose Blastomycosis. The urine is screened for antibodies against the B. dermatitidis yeast but unfortunately, this is not a perfect screening because it has been known to cause false negatives. Additionally, antigen testing can be done by drawing blood and isolating the serum to identifying the antigen. This test is much more reliable, however it is more difficult and time consuming to perform (Greene 2006).

Treatment for Blastomycosis consists of administering an antifungal medication until there are no signs of the yeast. Sterilizing an animal of B. dermatitidis is largely a waiting game. Itraconizole is the most effective drug to treat the infection because it rids the animal of the yeast the quickest (an average of 138 days). Other antifungals such as fluconazole are just as affective however the average time to do this is significantly longer due to the yeast’s production of melanin. When comparing costs, itraconizole is three times more expensive then the fluconazole. However, regardless of price, it may be more effective to treat the patient with an antifungal that can destroy cells faster and are not affected by the melanin (Mazepa 2011).

B. dermatitidis infects Midwestern dogs more than any other fungus and without affordable and effective treatments many die from Blastomycosis. Because there is largely no means of prevention, finding an accessible cure and reliable ways to diagnose patients is pivotal in the fight against the disease. Researching this organism could assist scientists in finding an effective cure which would save the lives of many pets in our own state.

By HZ

Works Cited:
Brömel, Catharina, and Jane E Sykes. 2005. “Epidemiology, diagnosis, and treatment of blastomycosis in dogs and cats.” Clinical Techniques in Small Animal Practice 20 (4) (November): 233-239.

Giles S, Klein B, Czuprynski C: The effect of canine macrophages on the adherence and growth of Blastomyces dermatitidis yeast: Evidence of a soluble factor that enhances the growth of B. dermatitidis yeast. Microb
Pathog 27:395-405, 1999

Greene, Craig E. 2006. Infectious diseases of the dog and cat. Saunders Elsevier, March 29.


Mazepa, A. S.W, L. A Trepanier, and D. S Foy. 2011. “Retrospective Comparison of the Efficacy of Fluconazole or Itraconazole for the Treatment of Systemic Blastomycosis in Dogs.” Journal of Veterinary Internal Medicine 25 (3) (May 1): 440-445.

The Power of Blue Cheese

When people hear the word penicillin, they most likely think of the antibiotic. Penicillin is a common antibacterial agent produced by the fungi Penicillium chrysogenum.¹ This particular genus, Penicillium, is also important for the production of several cheeses. A different strain, Penicillium roqueforti, is used to produce the characteristic flavor in blue cheese.² Like its antibiotic producing sibling, P. roqueforti creates unique compounds throughout its life cycle. The function of these compounds ranges from providing a tangy flavor to possibly preventing disease. Interestingly enough, blue cheese actually contains these beneficial compounds, such as andrastins and myophenolic acid.³ Although both molecules have very different functions, with andrastins displaying anticancer properties, and myophenolic acid used in organ transplant patients, their presence in blue cheese is a result of P. roqueforti. Less important than cancer prevention, other P. roqueforti products such as free fatty acids (FFA) and methyl ketones are essential in the production of blue cheese.² While blue cheese itself is not medically significant, it does provide an interesting environment to study the production of byproducts such as andrastins.

Figure 1: Blue Cheese

Blue cheese flavor comes from the mold spores of P. roqueforti, with the end product actually containing veins of mold. (Figure 1) The mold, which is actually a colony of fungus, plays an important role in developing the tangy flavor of the cheese. Just like human cells, fungi digest triglycerides into FFA, which are eventually reduced to methyl ketones.² The presence of FFA and methyl ketones gives blue cheese its distinct, tangy flavor. In order to achieve the ideal flavor, the cheese must have a good balance of FFA and methyl ketones, which is why P. roqueforti is used. P. roqueforti is very efficient at producing methyl ketones, and is used in the production of several blue cheeses including Roquefort. It is the ideal organism because as it grows in cheese, its ability to digest triglycerides increases exponentially. Like an acorn growing into a tree, fungi grow from a small seed like capsule called a spore, into a fruiting body called a mycelium. It's in this mycelium form that P. roqueforti can metabolize free fatty acids into methyl ketones at the ideal rate and produce a consistent flavor.²

The production of delicious blue cheese is not the only use for P. roqueforti, but it is definitely the most popular. Like its penicillin excreting sibling, P. roqueforti produces compounds that can be used medically. The most well-known compound is mycophenolic acid, which can be used during organ transplantations to prevent host rejection.³ As expected, mycophenolic acid can be found in blue cheese, as it is produced by P. roqueforti during growth. Thankfully, the effects of mycophenolic acid won't be observed after consumption of blue cheese, due to its low concentration, which is good news for avid consumers. The presence of myophenolic acid in blue cheese led to the inspection of blue cheese for other P. roqueforti products. This investigation led to the discovery of andrastins in several varieties of blue cheese cultured with P. roqueforti. Andrastins are a very unique molecule because they have only been found within blue cheese and within a laboratory environment.^3,4 (Figure 2) Currently, four andrastins have been identified, A, B, C and D. Andrastin A has garnered the most interest due to its presence in blue cheese and anticancer properties.  However, andrastins have not been shown to eliminate cancer within a patient, but they have been shown to have beneficial effects in laboratory experiments.

Unlike normal cells, cancerous cells divide uncontrollably, which results in tumors. The goal of most anticancer drugs is to prevent this unregulated multiplication, which is why andrastin A is of great interest. Andrastin A has been shown to help inhibit cellular division in cancerous cells, thus possibly slowing tumor growth.³ In addition to inhibiting growth, andrastin A has also been observed to aid in the accumulation of anticancer drugs in abnormal cells. Cancer cells have a unique surface protein that enables them to expel drugs and prevent their demise. Andrastin A is able to inhibit this protein from functioning and enable the drugs to accumulate and destroy the cancerous cells.³ The mechanism by which this occurs is not fully understood, but further research may lead to a new therapy for cancer patients. Further studies of andrastins themselves will also lead to new ideas about their role in the fight against cancer and if they posses other properties beneficially to human health.

The complexity of blue cheese goes far beyond its flavor. A unique organism, P. roqueforti, plays a huge role in the flavor of blue cheese and is also responsible for the production of several very important molecules. Free fatty acids and methyl ketones are produced by P. roqueforti< through the digestion of triglycerides, similarly to our cells after a high fat meal.² While these molecules are essential for the characteristic blue cheese flavor, they are not the most important molecules found in blue cheese. Myophenolic acid, a compound used during organ transplantation and andrastins, a newly discovered molecule with anticancer properties, are also produced by P. roqueforti.³ These molecules are both found in blue cheese, but are also produced by Penicillium strains in culture. Andrastins for example, were originally discovered in culture and later found to be present in blue cheese. The presence of these molecules in blue cheese is interesting because it means that humans have been consuming andrastins for hundreds of years. However, the concentrations of andrastins in cheeses are not currently known, but future research could determine any correlation between high consumption and cancer. It may be too soon to call blue cheese a “super food”, unless you're one who can't resist its tangy bite.

This piece of art submitted by Derek Hersch

References
1. Den Berg M Van, Gidijala L, Kiela J, Bovenberg R, Vander Keli I. Biosynthesis of active pharmaceuticals: β-lactam biosynthesis in filamentous fungi. Biotechnology & genetic engineering reviews. 2010;27:1-32. Available at: http://www.ncbi.nlm.nih.gov/pubmed/21415891
2. Kinsella JE, Hwang DH. Enzymes of Penicillium roqueforti involved in the biosynthesis of cheese flavor. CRC critical reviews in food science and nutrition. 1976;8(2):191-228. Available at: http://www.ncbi.nlm.nih.gov/pubmed/21770
3. Nielsen KF, Dalsgaard PW, Smedsgaard J, Larsen TO. Andrastins A-D, Penicillium roqueforti Metabolites consistently produced in blue-mold-ripened cheese. Journal of agricultural and food chemistry. 2005;53(8):2908-13. Available at: http://www.ncbi.nlm.nih.gov/pubmed/15826038
4. Omura S, Inokoshi J, Uchida R, et al. Andrastins A-C, new protein farnesyltransferase inhibitors produced by Penicillium sp. FO-3929. I. Producing strain, fermentation, isolation, and biological activities. The Journal of antibiotics. 1996;49(5):414-7. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8682716

Zombie Ants Go Marching

            Imagine a sudden insatiable need to climb shatters the monotony of your agonizingly redundant workday. As you climb higher and higher a sharp pulsating pain begins to emanate from the back of your head. Gaining in intensity which each thump, the pain has now become unbearable. Succumbing to the misery, with your last dying breath grasp onto the nearest structure within arms reach. After a period of time a fruiting body erupts from your corpse releasing spores hoping to find another victim. This scenario is a terrifying possibility for unlucky arthropods that have been selected as the specific host for pathogenic fungi belonging to the genus ophiocordyceps. One such relationship exists between the carpenter ant Camponotus leonardi and fungus Ophiocordyceps unilateralis

            Sensationalized through articles with titles such as “Fungus Makes Zombie Ants Do All the Work” and a feature in the incredibly popular documentary series Planet Earth, carpenter ants and the fungus have become the poster child for these particular host-parasite systems. What makes the fungus so interesting is the parasitic relationship in which the fungus has the ability to manipulate the behavior it’s host carpenter ant.

            The worker carpenter ants become infected when they come into contact with spores of the fungus. Contact is usually made on the forest floor and it is hypothesized that as a defense mechanism the ants only make the descent when there is no other way to traverse from plant to plant. The period of infection has been seen to be as short as 3-6 days.  Presumably as the fungus begins to establish itself within the ant’s carapace the behavioral modification sets in.

             By some unknown mechanism infected ants will voluntarily leave the colony and begin climbing as to not spread the fungal infection but it has also been observed that workers will actually carry out infected individuals unable to leave under their own power. As the infection worsens the ailing ant fastens itself at various places all over a plant but usually on the undersides of leaves.  Fastening is accomplished through a biting behavior seen as an extended phenotype of the fungus and will typically immediately precede death. The clenching of the ants mandible keeps the corpse in place as the fungal mycelia continue to propagate and also produce adhesives that more permanently bind the ant in place.

            Once the growing fungus is reproductively ready, a stroma will burst through the back of ant’s head.  The spore producing perithecia then grows from once side of the stroma.  Spores released by the fungus are too heavy to be wind dispersed and so just fall to the forest floor where they produce secondary spores that infect new hosts as they come into contact.

            What is remarkable is that large aggregates of dead ants have been observed in nature and deemed graveyards. In these graveyards researchers have seen as many as 14 dead ants per square meter transect.  During these research expeditions researchers also wanted to count how many live ants could be found in areas higher densities of dead ants. Despite extensive scouring of the areas of interest only 2 live worker ants were found compared to roughly 2500 dead ones.

            The carpenter ants were seen to only establish colonies in forest canopy with a network of aerial trails criss crossing through the trees. Very rarely were foraging trails seen traversing the forest floor.  As mentioned before, researcher hypothesized that the carpenter ants have developed this behavior of avoiding the forest floor and graveyards as a defensive mechanism against infection by fungus. Perhaps if a nearby but aerially inaccessible tree was seen to resourcefully profitable a colony would risk infection in order to colonize the tree.

            At the moment there are no pathogenic fungi like ophiocordyceps currently targeting humanity, but people should still maintain awareness. There could be a time when people you are close to become mindless zombies marching to their doom.

By Tuan Ngo

1) Graveyards on the move: the spatio-temporal distribution of dead ophiocordyceps-infected ants. Pontoppidan MB, Himaman W, Hywel-Jones NL, Boomsma JJ, Hughes DP. PLoS One. 2009;4(3):e4835. Epub 2009 Mar 12. PMID: 19279680

Investigation of the polar tube proteins of the Microsporidia parasite: Encephalitozoon cuniculi

The microsporidia parasite Encephalitozoon cuniculi is an intracellular parasite that is unique in that its complete genome is just under 2000 genes and 2.9 Mb in size (1). Microsporidia parasites infect all major mammal species, and are now one of the most common infections among immunocompromised humans. Infections of this parasite are transmitted by internalization of spores via the respiratory system or gastrointestinal tracks. This organism can survive outside of a host for several years as dormant microscopic spores that range in size from 1 to 40 μm. The spores are covered in a thick double wall composed of a chitin-rich endospore and a protein based exospores layer. Within the double wall is the sporoplasm that contains the nucleus and cytoplasm of the microsporidia along with the polar tube organelle required for the infection process, and transfer of the sporoplasm into the host cells (2).

This defining trait of the microsporidia phylum is the polar tube that is wrapped tightly around the periphery of the sporoplasm until time of spore germination, yet its polar tube protein structure is still not well understood. E. cuniculi became the model microsporidia organism when the organism’s full genome was successfully sequenced. The polar tube of E. cuniculi is a proteinaceous structure composed of three separate proteins: polar tube protein 1 (PTP1), PTP2, and PTP3 (2). These three proteins share little sequence homology with one another or with the BLAST or PFAM databases. This suggests that these proteins are specific to microsporidia and evolved during the transition from fungal like cells to intracellular parasites (2).  Following an external signal for spore germination, the contents within the endospore begin to swell and pressure begins to build. This swelling occurs due to a rapid influx of water across aquaporin channels in the plasma membrane (2). After the spore wall reaches its maximum threshold for pressure build up, the anterior end of the spore wall ruptures and the polar tube extends as the sporoplasm is forced out of the spore and down the tube. It is hypothesized that the the central core PTP1 of the polar tube binds to the host cell membrane and then through endocytosis of the polar tube, the sporoplasm enters the host cell (2).

Recently research was conducted to investigate the interaction of the polar tube proteins of E. cuniculi to better understand the formation of the polar tube. An understanding of the protein structure of the polar tube is crucial in developing methods to prevent microsporidia infection and subsequent disease caused by the microbe. The three distinct polar tube proteins were the focus of the study: PTP1 that is rich in proline protein, PTP2 that is rich in lysine protein, and PTP3 that is uniquely larger than the other two polar tube proteins. The preservation of cysteine residues in PTP1 suggest that it may be involved in intraprotein or interprotein linking leading to the formation of the polar tube. The study was focused on the interactions between the major protein PTP1 with itself and the other polar tube proteins (3).

In order to investigate where the PTP associated on the polar tube, E. cuniculi PTP1, PTP2 and PTP3 were expressed as fusion proteins and then from these recombinant proteins were used to prepare polyclonal antibodies. The accuracy of the antisera prepared for each polar tube protein was confirmed on an immunoblot analysis. Each corresponding antisera was able to detect its corresponding polar tube protein from the E. cuniculi lysate. In addition, all of the antisera, anti-EcPTP2, anti-EcPTP2, and anti-EcPTP3, reacted with the E. cuniculi polar tube by indirect immunofluoresence assay and viewed under fluorescence microscopy (3).  The staining of the polar tube with anti-EcPTP2 covered the entire structure with green staining and the staining of the polar tube with the anti-EcPTP3 also covered the entire structure of the polar tube with red staining. When these two images were overlaid the image was yellow which suggested that the polar tube proteins overlap within the polar tube and are not isolated to specific locations (Figure 1). This immunofluoresence assay was repeated with anti-EcPTP1 and anti-EcPTP2, and resulted in the same type of overlap of the polar tube proteins on the polar tube (3).  Also, it’s important to note that none of the antisera reacted with surfaces of nongerminated spores.

Figure 1. Immunofluorescent analysis of PTP antibodies.
Images of extruded polar tubes of E. cuniculi that were incubated
with one of the three polar tube antibodies: anti-EcPTP1,
anti-EcPTP2, and anti-EcPTP3. The polar tubes were then
labeled with a second polar tube antibody and a fluorescent
label. (A)The polar tube was incubated with anti-EcPTP1 and
a green fluorescent marker. (B) Polar tube incubated with
anti-EcPTP3 with fluorescent red. (C) Merged image of image
A and B. (D). Polar tube incubated with anti-EcPTP2 and a
green fluorescent marker. (E). Polar tube with anti-EcPTP3 and
red fluorescent marker. (F) Merged image of image D and E.
(G) anti-EcPTP1 with green fluorescent marker. (H) anti-EcPTP2
with red fluorescent marker. (I). Merged image of G and H.

In order to investigate the possibility of an interaction among the three different PTPs a yeast two hybrid analysis was conducted. In these experiments, the E. cuniculi PTPs were analyzed in all possible pair-wise combinations; fused either to the bait or prey vector (3). A two hybrid yeast analysis consists of amplifying the PTP gene by PCR amplification, integrating it into a plasmid vector, and then transforming yeast cells with both the bait and prey vectors. The growth of the transformed yeast colonies was observed as evidence of the in vivo interaction of the two polar proteins. The researchers show that growth of all the yeast cells containing the different PTP combination suggest that all three of the E. cuniculi PTP can interact with themselves and each other, although the domain responsible for this interaction remains to be determined. Another two-hybrid yeast analysis focused on the compatibility of the N- and C- terminal regions of PTP1 provided a similar results that suggested that both the N- and C- terminal regions of PTP1 can interact with each other and themselves (3). 

Overall, this study makes a first attempt to better understand how the proteins of the polar tube of the microsporidia E. cuniculi are associated to gain insight to how the polar tube functions. This provides the first steps into more research of understanding how the proteins interact to produce the invasive polar tube that is used by microsporidia for infection, and thereby gaining a perspective how to prevent this pathogen from infection and prolonged disease.

Submitted by KB

Works Cited

1. Sibley, L. D. Invasion and intracellular survival by protozoan parasites. Immunological Reviews, 2011. (240) p.72-91.

2. Williams, Bryony. Unique physiology of host-parasite interactions in microsporidia infections. Cellular Microbiology, 2009. (11):11. p. 1551-1560.

3.  Bouzahzah, Boumediene, et. al., Interactions of Encephalitozoon cuniculi polar tube proteins, 2010. (78):6 p. 2745-2753.