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).

Entamoeba histolytica: A Dangerous Stowaway

Figure 1:  A histological slide of amoeba infected brain tissue.
The left side of the image shows a trophozite that has disseminated
to the brain tissue.  The smaller darker spots in are amoebic cysts
that have formed in the brain tissue.

            Amebiasis is an infection of the intestines that is caused by the parasite Entamoeba histolytica.  This parasitic amoeba infects the host through the fecal-oral rout.  However the parasite may spread and infect other organs such as the liver and, or the brain.  Infection of the brain can be fatal (figure 1 [1]). Infection by E. histolytica happens when the host 1).  Ingests a mature cyst containing the parasite, found in food, water, or on unwashed hands that are contaminated with fecal matter.  2). Excystation of the mature cyst (where the parasite escapes from the cyst) occurs in the small intestine.  3). Trophozoites (the active and motile stage of the parasite) are then released and migrate to the large intestine. The trophozoites multiply by binary fission and produce cysts.  4). These cysts are then passed in the feces of the host.  Because of the protection conferred by their walls, the cysts can survive days to weeks in the external environment and are responsible for transmission.  Trophozites can also be transmitted through fecal matter as well, but do not last long in the environment and are destroyed quickly. (Figure 2 [2]). 

Figure 2:  A diagram of E. histolytica life cycle in the human body,
including possible sites where the parasite can disseminate to cause disease.    

However In many cases the trophozoites remain confined to the intestinal lumen of individuals who are thus asymptomatic carriers, and cyst passers. This is a non-invasive infection.  In some patients the trophozoites invade the intestinal mucosa and cause intestinal disease, like colitis.  The parasites can also disseminate in the body through the bloodstream, invading extra-intestinal sites such as the liver, brain, and lungs causing extra-intestinal disease.  The result of which can lead to sever pain in certain regions of the body, liver abscess, high fever, and even death. Transmission can also occur during sexual contact, in which not only cysts, but also trophozoites can be transmitted.  All sexually active individuals who practice unsafe oral-anal sex, and immunocompromised individuals are at an increased risk for contracting an E. histolytica infection.

In a study by Salit et al. researchers found a cluster of E. histolytica infections in Canada.  They traced this cluster to several individuals.  Patient A who is a female, patient B who is a female, and patient C who is a male, had all recently traveled to England, Germany and Southern Italy together and returned on September 2007.  Five months later patient A developed anorexia, chills, a fever of 39.5º C, and abdominal pain.  After being admitted to the hospital, she was found to have a liver abscess (an accumulation of puss) and colitis (inflammation of the intestines).  After several tests, it was discovered that she had been infected with a highly virulent strain of E. histolytica that had disseminated and caused her liver abscess.  Three months after, patient C was also found to have two liver abscesses in the same part of the liver as patient A.  The tests concluded that he too was infected with the same highly virulent strain of E. histolytica.  Patient B was infected with the same strain as well, and had developed lymphadenopathy (refers to enlarged lymph nodes).

The researchers found that all three of these patients had been sexually active together, and had other sexual partners who were labeled patients D, F, and G.  Patient A had another sexual partner, patient E, who also was diagnosed with a liver abscess and intestinal colitis as well.  Patients D, F, and G were female homosexuals and bisexuals who were sexually linked to patients A, B, and C.  They all reported engaging in oral-anal sexual activity.  Patients D, F, and G were also found to be infected with E. histolytica but they did not show any symptoms.  It was concluded that although patient E did in fact travel to Iraq and Africa, it was most likely that he contracted the infection from engaging in sexual activity with patient A. [5] These cases are an example of how this parasite can be very easily transmitted from one person to another.  The patients in this study all practiced oral-anal sex with each other, which lead to an increased chance of contracting the parasite from the infected partner.

It is known that individuals with compromised immune system; are more often infected with common microorganisms that would usually not be deadly, individuals infected with HIV-1 for example.  However a recent study by Nagata et al. had some interesting finding when comparing risk factors for amebiasis infections.  They conducted a study on 10,930 patients infected with amebic colitis over a span of seven years.  They studied the infection pattern in Japan from 2003 to 2009 they found that there was a significant increase in the number of infections in this time span.  They performed endoscopic tests on all the patients at that time and confirmed amebic colitis.  The researchers found that in 2003 there were about 3 out of every 1,904 patients that had tested positive for amebic colitis, whereas in 2009 there were 12 in every 1,360 patients that had tested positive.  That is a 5.6-fold increase in the number of cases of colitis related to amebic infections in the span of seven years.

The researchers assessed the risk factors associated with this jump in infections, and concluded that were several factors that can be traced back to individuals who were HIV-1 positive, individuals who had a history of syphilis, and individuals who were sexual activity with commercial sex worker (CSWs).  However when they investigated further into the HIV-1 risk factor, they found that there was no association with the infection itself and being HIV-1 positive, rather it was the risky behavior that these individuals engaged in that led to their higher rate of infection. [4]

            Amebiasis is a serious condition that is caused by Entamoeba histolytica.  It is not a major cause for concern in most industrialized countries.  However when contracted it can be dangerous and sometimes fatal if not treated quickly.  It can cause liver abscess, colitis, and if disseminated to the brain can cause death.  Everybody can get this infection, although sexually active individuals, especially those individuals who practice unsafe oral-anal sex, have to be cautious and take enteric precautions (precautions against possible contamination from bodily fluids of the gastrointestinal (GI) tract of a person suspected of having a GI infection) to keep from getting infected with this parasite.  Also immunosuppressed individuals have to be careful of this infection, as well as people who do not have access to adequate hygiene or a clean water source.

Reference:

1.     "Amebic Brain Abscess: MedlinePlus Medical Encyclopedia Image." U.S National Library of Medicine. U.S. National Library of Medicine. Web. 17 Nov. 2014. http://www.nlm.nih.gov/medlineplus/ency/imagepages/1047.htm.

2.     "Amebiasis." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention. Web. 3 Dec. 2014. <http://www.cdc.gov/dpdx/amebiasis/>.

3.     "Amebiasis Life Cycle." Amebiasis Life Cycle. Web. 17 Nov. 2014. <http://www.pharmacology2000.com/Chemotherapy/Antiparasitic/Amebiasis_Life_Cycle1.htm>.

4.     Nagata, Naoyoshi, Takuro Shimbo, Junichi Akiyama, Ryo Nakashima, So Nishimura, Tomoyuki Yada, Koji Watanabe, Shinichi Oka, and Naomi Uemura. "Risk Factors for Intestinal Invasive Amebiasis in Japan, 2003–2009." Emerging Infectious Diseases: 717-24. Print.

  

5.     Salit, Irving E., Krishna Khairnar, Kevin Gough, and Dylan R. Pillai. "A Possible Cluster of Sexually Transmitted Entamoeba Histolytica: Genetic Analysis of a Highly Virulent Strain." Oxford Journals (2009): 346-53. Print.