Fungi That Will Make Your Skin Crawl

by Lev Ostrer

An interesting and unexpected discovery was made in 1998 about a Chytridiomycota family of fungi that had taken scientists by storm. Prior to this discovery the Chitrid family was believed to be a family of decomposing fungi that lived on dead animals and other decomposing matter(1), but this was all about to change with a discovery of a new family member, Batrachochytrium dendrobatidis. Unlike the rest of the family, B. dendrobatidis prefers to live inside living things. Primarily it lives inside amphibian skin of frogs, toads and even salamanders. This clever fungus figured out a way to get under the skin and live there while spitting out large amounts of motile zoospores, which infect more amphibians that share the same water source. The incredibly mobile zoospores can also be picked up by birds and animals and moved to other bodies of water. Though might be a great survival strategy for the fungus many species of amphibians die due to the fungus thriving under their skin.

Fig 1. 2 types of cells in the frogs’ epithelium, principal and a
mitochondria cell. A principal cell is using pores to uptake water,

 and ATPase to move K+ from plasma and Na+ into the plasma.

Mitochondria rich cell uses pores to uptake Cl- and ATPase

 to K+ and Na+.

For many organisms that have skin, skin’s primary job is to keep unwanted things out and to keep the insides safe; however for frogs, skin is much more than a protective barrier. For frogs skin is also used as a way to keep hydrated and a way to breathe. Up to 90% of the frog’s epidermal surface is made up of cells called principal cells (1). These cells play an important role in electrolyte and water transport using a variety of channels and ATPases (transport proteins that use ATP to move molecules against chemical/concentration gradient) to achieve proper K+ and Na+ concentration within the organism (fig1). The other 10% of epidermal cells are mitochondria rich cells that are also involved in transport of electrolytes, but these cells specialize in Na+ and CL- transport. When Batrachochytrium dendrobatidis invades the skin, it inserts itself under the top layer of epidermis and begins producing keratin. Keratin is the main component of hair and nails, so in effect the infected frog has hair/nail like material growing under its skin. The worst part is that this causes the top layer of skin to peel off (fig2), which in turn causes a frog to experience reduction of Na+ absorption, as well as, a drop of K+ and Cl- in plasma. This will eventually result in a heart attack due to skewed electrical gradient (2). While K+ and Cl- levels are falling, during late stages of infection, a frog is also experiencing extreme dehydration, which cause amphibian to spend more time near water thus releasing more zoospores into environment. The infection is directly related to the fungus life cycle, where keratin is made in order to promote zoospore release.

Fig2. L. catesbeinus 24 hours post infection; top image shows

 a premature keratanization (the dark layer of skin) with major

 infection sites indicated by large black arrows, and small white

 arrows showing the location of zoosporangia. The bottom image

 shows a infection after 60 hours, when skin begins to come off.

 Black arrows are pointing at keratin deposits and small

 arrows are showing the location of zoosporangia.

Batrachochytrium dendrobatidis has an interesting life cycle that can be broken down in two phases; Substrate-independent (a free swimming zoospore) and substrate-dependent (where it invades a host) (fig3). A young zoospore is equipped with flagella, and after leaving zoosporangium it has about 24 hours to attach to a host. Once it attaches several changes take place. First it loses its motility by retracting its flagella, and begins making a wall of chitin around the spore. At the same time a germination tube begins to form, which penetrates the top epithelial layer and injects it’s contains into a host. This causes a formation of a cyst under the skin of the frog. The cyst stays in the deeper skin tissue until it matures. A mature cyst then begins producing keratin in order to form a barrier to separate zoosporangium from the germination tube. Due to the loss of skin cells, a host frog begins to make more skin cells, causing a mature cyst to move outwards, towards the newly formed epithelial layer (2). Once zoosporangium (cyst) reaches the surface a plug on the surface of the cyst gets removed and new generation of motile zoospores leaves ready to infect nearby cells as well as other amphibian inhabitants of the ecosystem (fig3). As more cells become infected, keratin production goes up, eventually causing chunks of skin to fall off (fig2), finally resulting in the host’s death.

Fig3. A life cycle of Batrachochytrium dendrobatidis starting with
 motile zoospores on top , and going counter clockwise thought
attachment/ host infection ( left images) , onto cyst maturation
 ( two bottom images) and lastly plug removal and zoospore
 release ( right side).

However the story doesn’t end here; frogs are finding clever ways of fighting back the fungus. Many neotropical species of steam-dwelling harlequin toads and frogs such as A. elegans that live in lowlands at 25C° are beginning to show a great amount of resistance against the B. dendrobatidis. In part, this is due to the high temperature of 25 C° at which fungus grows slower allowing for more time to mount an immune response before full spread infection. This is not surprising because a life cycle of Bd, is very temperature sensitive, where fungus thrives between 4-23C°, and it dies when temperature is over 28C °or under 4C°.The colder temperature gets in its healthy range, the longer it takes for cyst to mature, and the warmer it is the faster zoospores are produced (4). On top of temperature being in the frogs’ favor, harlequin toads and frogs were found to grow their own antifungal medication (3). Scientist went out and swabbed the skin of 3 different members of the Atelopus (harlequin toads and frogs common to Central and South America) family that showed an increased resistance to Bd. Of 148 swabbed bacterial species isolated 26% turned out to have antifungal properties. One particular species of frogs stood out by far from all the others based on its ability to fight off the fungus.40%of bacteria found on A.elegans skin had antifungal activity. This species, interestingly enough was also the only species that tested positive for the fungus. This indicated that A. elegans had undergone a natural selection event, where members of the species without antifungal bacteria died off, and ones that remained had an increased amount of antifungal bacteria on its skin.

Thus the battle continues, between parasitic fungi and frogs. Hopefully it soon will come to the end since many amphibian species that are dying from the infection are in danger of going extinct. Fortunately species like A.elegans are showing the way this fungus can be slowed, if not stopped.  Maybe endangered frogs can take a lesson from A. elegans, and if needed with a little help from humans harness the power of antifungal bacteria to prevent extinction. 

References:

1. Craig R. Campbell, Jamie Voyles, David I. Cook, Anuwat Dinudom., Frog skin epithelium: Electrolyte transport and chytridiomycosis. The International Journal of Biochemistry& Cell Biology, 44:431-434, (2012)
2.Sasha E. Greenspan, Joyce E. Longcore, Aram J. K. Calhoun., Host invasion by Batrachochytrium dendrobatidis: fungal and epidermal ultrastructure in model anurans. Disease of Aquatic Organisms, Vol. 100: 201–210, (2012)
Surviving Chytridiomycosis: Differential Anti-Batrachochytrium dendrobatidis Activity in Bacterial Isolates from Three Lowland Species of Atelopus.

PLoS One. 7(9): e44832, (2012)
3. Sandra V. Flechas,Carolina Sarmiento,Martha E. Cárdenas, Edgar M. Medina, Silvia Restrepo, and Adolfo Amézquita., Surviving Chytridiomycosis: Differential Anti-Batrachochytrium dendrobatidis Activity in Bacterial Isolates from Three Lowland Species of Atelopus. PLoS One. 7(9): e44832, (2012)
4. BUSTAMANTE H, LIVO L, CAREY C., Effects of temperature and hydric environment on survival of the Panamanian Golden Frog infected with a pathogenic chytrid fungus. Integrative Zoology [serial online]. June 2010;5(2):143-153

Organism: Entamoeba histolytica

by GP

Entamoeba histolytica is an anaerobic protozoan parasite that primarily infects digestive tract in humans and primates.  This pathogenic microorganism is the fourth leading cause of deaths and third leading cause of morbidity due to protozoan infections worldwide (1).  Transmission of E. histolytica occurs when a person ingests food or water that is contaminated with infected feces or during sexual intercourse (2).  This sort of transmission is more prevalent in developing countries with poor sanitary conditions.  The infection of E. histolytica is usually called amebiasis or amoebiasis (1,2).  Amebiasis occurs in the large intestines, which causes an internal inflammation. The internal inflammation is caused by trophozoites, which are infected cells that enter the intestines. The trophozoites come into contact with human cells, which induce a rapid influx of calcium in the cell.  This causes all membrane movement to stop.  The internal workings of the cell are disrupted, organelles lyse and, cells die. Consequently, the amoeba vacuums up the dead cell.

Figure 1. The Life Cycle

of E. histolytica

The course of infection for this parasite starts with mature cysts entering the human body, by ingesting mature cysts in fecally contaminated food or water.  Once inside the body, trophozoites develop in the large intestine and produce cysts. In some cases, the trophozotic cysts could remain undetected in the intestinal lumen and pass through by feces.  The cysts that exit the body by feces in cyst form can survive outside the body for several weeks.  In other cases, the trophozoites can pass the mucosal barrier or enter the bloodstream that causes the infection to occur (2, Figure 1). The symptoms of the disease include diarrhea and abdominal pain for mild cases.  For severe cases of the disease the symptoms may include stomach pain, blood and mucus in feces, and high fever (2).

Figure 2. Entamoeba histolytica with four nuclei

Often E. histolytica is misdiagnosed or mistaken for other similar infections.  When diagnosing amebiasis, stool samples are usually taken and it is difficult to distinguish if the infection is caused by E. histolytica due to the similarity to other infections and its symptoms as well.  However, a way to determine the presence of the parasite is to examine the number of nuclei (Figure 2). For example, the mature cysts have four nuclei, which is a telltale sign of Entamoeba histolytica cysts. Other than stool samples, enzyme-linked immunosorbent assay (ELISA) can be used to evaluate the sensitivity level of the parasite in cells.  ELISA is a useful tool in medicine to measure the interaction between antibodies and antigens from foreign microorganisms. ELISA is used to examine the trophozoites because at this stage in the cell cycle of Entamoeba histolytica, they are in the intestinal tract and can be easily extracted than cysts from stool samples.  The trophozoites are treated with different detection kits for ELISA and the level of expression of each kit are further analyzed in PCR, and this helps to know which substrate and substance identify the pathogenic parasite.  Thus, knowing which enzyme substrate and serum substances to use for identifying Entamoeba histolytica, the diagnosis of the disease, amebiasis, cannot be confused by other pathogens that share similar characteristics.  Stool samples examined under microscopy only show that parasites are present, but cannot tell what type of parasite it is.  Treating amebiasis with proper diagnosis and antibiotics, such as auranofin, can save lives and inform the public about how the parasite spreads. Diagnostic steps can be taken, like using stool samples and ELISA, to ensure the right diagnosis is made.

After diagnosing the patient with amebiasis, there are several treatments for Entamoeba histolytic, but the rise of antibiotic resistance is a major concern.  The treatment of amebiasis involved metronidazole, which E. histolytica is now resistant to (1) (Oh NO!).  Have no fear; new drugs are being made to target specific sites of amebiasis.  Drug screen tests have identified auranofin as an effective drug against the parasite.  This drug is originally used for treating rheumatoid arthritis.  To understand how the drug is targeting E. histolytica, transcriptional profiling and thioredoxin reductase assays show the drug targets the parasite’s thioredoxin reductase.   Thioredoxin is a protein that acts like an antioxidant and helps reduce other proteins by cysteine thiol-disulfide exchange. This is an important biological process because preventing thioredoxin reduction in E. histolytica means disulfide bonds in cells are not reduced.  Since the disulfide bonds are not reduced, sensitivity to reactive oxygen species (ROS) is increased in which cell structures can be damaged (1).

Along with disrupting thioredoxin reductase, the role of adherence to mucosal membrane is another target site for drugs (3).  As mentioned above, the course of infection mainly pertains to the large intestines, thus the parasite seems to adhere and degrade the mucosal barriers.  Research by Ravdin et al. found that the adherence of E. histolytica to target cells requires microfilament function, which lyse cells, and has a specific amebic receptor that is attracted to N-acetyl-d-galactosamine (GALNAc) (3).  The adherence of amebas to a functional group, CHO, on cells show carbohydrate specificity and it is a key component of the contact-mediated killing of target cells.  GALNAc inhibits the amebic cytolysis of target cells, so cells cannot be killed.  Since the cells cannot be killed, the parasite cannot adhere to the mucosal barriers and cannot enter the bloodstream (3,4).  To target the adherence site and adherence receptors on Entamoeba histolytica and cell surface, respectively, drug therapies can be made to specifically target that region, like using GALNAc.

All the aspects of Entamoeba histolytica is known in terms of being the first human amoeba to have its genome sequenced and analyzed.  The Sanger Institute is the first to sequence the genome and with their help, further insight of the amoeba parasite can provide evolutionary DNA markers and mechanisms of infection.  The human amoeba parasite, Entamoeba histolytica, infects 50 million people around world and 70,000 deaths occur.  All hands are on deck to fight Entamoeba histolytica and prevent misdiagnosis.

Citations

1. Sharon L Reed, et al. "A High-Throughput Drug Screen For Entamoeba Histolytica Identifies A New Lead And Target." Nature Medicine 18.6 (2012): 956-960. Academic Search Premier. Web. 15 Nov. 2012.
2. Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 02 Nov. 2010. Web. 15 Nov. 2012. 
3. Ravdin, Jonathan I., and Richard L. Guerrant. "Role of Adherence in Cytopathogenic Mechanisms of Entamoeba Histolytica." Journal of Clinical Investigation 68.5 (1981): 1305-313. Print.
4. Barbara J. Mann, et al. "Identification Of Entamoeba Histolytica Thiol-Specific Antioxidant As A Galnac Lectin-Associated Protein." Molecular & Biochemical Parasitology 127.2 (2003): 113. Academic Search Premier. Web. 15 Nov. 2012.
5. Mirelman, David. "Comparison of Use of Enzyme-linked Immunosorbent Assay-based Kits and PCR Amplification of RRNA Genes for Simultaneous Detection of Entameba Histolytic and E. Dispart." Journal of Clinical Microbiology 35.9 (1997): 2405-407. Print.