Friday, December 12, 2014

Drug Resistance: The Arms Race Between Trypanosoma brucei and Arsenical Drugs

By Savannah Kaye

Drug resistant infections have become a major problem in modern day medicine. Humans develop drugs to fight an organism. The organism responds by developing counter measures to block the drugs, rendering them useless. This back and forth adaptation causes an arms race between humans and organisms, as each tries to gain an advantage and become the victor.  Arsenical drugs work by taking advantage of an amino-purine transporter in Trypanosoma brucei to get the drugs into the cell (1). Melarsoprol B is a melaminophenyl arsenical drug whose mechanism of action is not fully understood. It is known to get into the cells using P2, an amino-purine transporter also called TbAT1, and inbibits glycolytic enzymes, phosphogluconate dehydrogenase, and trypanothione reductase (2). Trypanosoma brucei is a protozoan parasite that is the causative agent of African Sleeping Sickness in humans, as well as other diseases in various species. Trypanosoma brucei has developed a way to prevent arsenical drugs from killing the parasite. One such mechanism makes use of the adenosine transport system (3). T. brucei has a mutation in the adenosine transporter that prevents the cells to take up both exogenous adenosine and arsenical drugs. The evolution of drug resistance has started an arms race between humans and pathogens that shows no signs of being resolved.

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Trypanosoma brucei comes in three different varieties, which can infect both animals and humans. T. brucei brucei causes Nagana in cattle (4). The two varieties that cause African Sleeping Sickness in humans are T. brucei gambiense and T. brucei rhodsiense. T. brucei gambiense is responsible for roughly 98% of T. brucei infections and found in more than 24 countries in West and central Africa (4). T. brucei gambiense, causes a chronic infection, in which symptoms do not manifest for months to years after infection. T. brucei rhodsiense, on the other hand, makes up the last two percent of T. brucei infections and is limited to thirteen countries in Eastern and Southern Africa. This type of infection is more acute and symptoms develop weeks to months after infection(4). African Sleeping Sickness, or African Trypanosomiasis, symptoms can be broken down into two stages: stage one symptoms and stage two symptoms. Stage one symptoms are characterized by fever, headache, weakness, itching, and joint pain. Treating African Sleeping Sickness after the manifestation of first stage symptoms is effective, however it is difficult to diagnose from first stage symptoms (5). Second stage symptoms are more severe; symptoms include convulsions, confusion, and violent behavior. One other interesting  symptom of African Trypanosomiasis is that patients are often unable to sleep at night, yet become overwhelmed by sleepiness during the day (5). Arsenical drugs are used as second stage treatments (4). Since these drugs are more toxic and can cross the blood brain barrier, they are reserved for more severe symptoms(4). Some common examples of arsenical drugs are Melarsoprol and Trypursamide(6). These drugs are extremely toxic and are only used when the infection has spread to the central nervous system. Because of the late detection of Trypanosomiasis, the parasite has established a strong hold on the host. The strongly established growth in the host makes it so that the drugs create a selective environment where the resistant strains are able to recolonize the host once the competition is eliminated.

African sleeping sickness is difficult to treat since some strains of T. brucei have developed mechanisms for resistance. These strains have a mutation in the gene that encodes an adenosine transporter which prevents cells from pumping arsenicals into the cell. To test the role of adenosine transporters in arsenical resistance, genes for TbAT1 from both susceptible and resistant strains of T. brucei brucei were cloned into purine auxotrophic Saccharomyces cerevisiae. The yeast, like T. brucei, are unable to make their own adenosine and therefore cannot survive unless they pump adenosine into the cell (3). This experiment makes use of the characteristic nature of yeast, which do not normally take up exogenous adenosine. After yeast cells were transformed with the gene for TbAT1 from T. brucei brucei, they were grown on plates containing adenosine, which the cells normally are unable to take up, and adenine, which cells readily take up. Next, the amount of adenosine and adenine transported into the cell was measured. Resistant strains and control yeast were not able to take up adenosine and were not able to grow when plated on 150 μM adenosine concentrations.

There are two types of adenosine transporters, called P1 and P2(3), which differ in specificity. P1 transporters are specific for adenosine and inosine. P2 transporters are specific for adenosine, adenine, and melaminophenyl arsenicals (3). In this study, they tested the inhibition of adenine transport in the presence of inosine and other arsenicals. Adenosine transport was not inhibited in the presence of inosine, indicating that inosine was not the substrate of the adenosine transport pump. Melarsoprol, melarsen oxide, and isometamidium are all substrates for the adenosine transporter and in the presence of each of these arsenical drugs, adenosine transport into the cell is inhibited, indicating that the substrate of the pump was adenosine and arsenical drugs not adenosine and inosine. Therefore, the pump exhibits P2 activity, not P1 activity (3). While the genes used were from T. brucei brucei, the results can be translated to other subspecies of T. brucei.

The war between humans and pathogens rages on, as each species fights for life. Parasitologists study these parasites so they can understand how the parasites develop resistance to drugs. By understanding the mechanisms of drug resistance, scientists can develop other drugs that will fight these infections. Just when they have figured out the mechanism of drug resistance and start treating the infection, the parasite finds a new mechanism of drug resistance. The parasites must respond to the development of new drugs by building up their arsenal and developing novel ways to resist being killed by a new drug. They invoke many different strategies to prevent pumping the drugs into the cell or by creating new enzymes to break down drug components. This fight between humans to kill pathogens and parasites to fight drugs has led to an arms race between humans and parasites that continues to escalate with no end in sight.

Works Cited

1. H. Denise, M. P. Barrett, Uptake and mode of action of drugs used against sleeping sickness, Biochemical Pharmacology 61, 1–5 (2001).

2. M. E. Schweingruber, The melaminophenyl arsenicals melarsoprol and melarsen oxide interfere with thiamine metabolism in the fission yeast Schizosaccharomyces pombe., Antimicrobial agents and chemotherapy 48, 3268–71 (2004).


3. P. Mäser, A Nucleoside Transporter from Trypanosoma brucei Involved in Drug Resistance, Science 285, 242–244 (1999).


4. World Health Organization, Trypanosomiasis (available at http://www.who.int/mediacentre/factsheets/fs259/en/).


5. Medecins Sans Frontiers, Sleeping Sickness (available at http://www.doctorswithoutborders.org/our-work/medical-issues/sleeping-sickness).


6. Trypanosoma brucei (available at http://parasite.org.au/para-site/text/brucei-text.html).



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