The role of nucleotide transporters in host-parasite interactions: A look at drug resistance and efficacy in Trypanosomiasis.

 

By April D. Adrian

(figures will be added later)

African Trypanosomiasis is a devastating disease that plagues the health, productivity and prosperity of thousands of human lives each year.  The fly belt of Africa (15⁰N to 25⁰S) is home to trypanosomiasis and nagana.  Trypanosoma brucei brucei infects native antelope and introduced livestock and is referred to as nagana.  It does not cause disease in humans. There are two forms of trypanosomiasis: East African Trypanosomiasis, (EAT), and West African Trypanosomiasis, (WAT), both caused by a vector-borne parasitic protozoan of the genus Trypanosoma.  Trypanosomiasis results from the painful bite of a tsetse fly that harbors the infective metacyclic trypomastigotes of T. brucei rhodesiense or T. brucei gambiense. The protozoan then replicates in the blood and lymph of its vertebrate host.  T. brucei rhodesiense (EAT) causes acute infection and T. brucei gambiense (WAT) causes chronic infections.  The parasites invade interstitial spaces of the spleen, central nervous system and the brain (T. brucei gambiense only). Symptoms include: intermittent fever, swollen lymph glands, generalized pain, weakness, headache and cramps. T.b. rhodesiense infection results in rapid weight loss and heart problems.  T.b. gambiense causes abnormal fatigue, mental dullness, apathy, disturbances of coordination, and uncontrollable sleepiness. These symptoms are responsible for the common name given to this disease, African Sleeping Sickness.  Paralysis, convulsions, coma and death are common outcomes in untreated cases (Roe, 2001). 

            The treatment of Trypanosomiasis has had a rather unsuccessful history.  Arsenical drugs were the first attempt at treatment, but they came with several drawbacks.  Arsenic causes eye damage in high doses and is best administered intravenously.  The trypanosomes quickly mounted resistance to the first line of defense.  A new class of arsenicals, melaminyl-substituted phenylarsonates, are derivatives of some of these originals,(i.e. melarsen oxide).  Melarsoprol, (fig 1), was brought into the line up in the 1940’s and is effective in the late stages of the disease because of its ability to cross the blood brain barrier. Although Melarsoprol is still in use today,  it has potentially fatal side effects that make it impossible to use outside of a continuous care facility. Melarsoprol works by reacting with sulphydral groups on  enzymes and various other substances in the parasite as well as the host.  Drug uptake is thought to be based on the permeability of cells.  It is thought that the mammalian cell may not be as permeable to the drug as the trypanosome cell and this may account for the increased sensitivity of the parasite to the drug, (Harvey, 1997).  It has been proposed for some time that resistance to this drug may be due to the decreased permeability of trypanosome cells, (Roberts 1989).  The second line of defense, (suramin, pentamidine, and others), is somewhat successful in the treatment of early cases but is ineffective in cases involving the central nervous system. Pentamidine, (fig 2), has two very polar amidino groups on either end making it incapable of passing through the blood brain barrier. Likewise, Suramin, (fig 3), is a sodium sulfite salt and thus does not cross the blood brain barrier.  Pentamidine and Suramin are still in use today but there is evidence of resistance to both drugs.   One of the more recent treatments for CNS infections is diflouromethylornithine (DFMO). DMFO treatments increase the rate of host survival and even results in some apparent cures.   It is currently the drug of choice even though some strains of the parasite are innately resistant to its effects (Roberts, 2000).

The rise of drug resistance, and the increasing need for new chemical therapeutic agents is driving research on trypanosomiasis.  In the past decade researchers have uncovered clues that point to the molecular basis for the delivery of chemical therapeutics into the parasite, and are continuing their studies to elucidate possible ties to the development of resistance. The future of trypanosomal therapy may lie in a molecular dissection of the parasites’ nucleotide transporters, (NTs). This new field has a number of pathways to be explored.  Molecular clones and expression systems offer the tools needed to access this information.  Knowledge in this area could help treat the 20,000 new cases of trypanosomiasis that arise each year, and in addition the principles of this type of study could be applied to most parasitic protozoans.

 

Most protozoan parasites, including Trypanosomes, are incapable of synthesizing nucleotides on there own. They must acquire ucleotides by transporting preformed host nucleotides across membranes with nucleotide transporters.  The differences in host and parasite nucleotide transport may offer a new avenue for therapeutic action.  The two known NTs in Tyrpanosoma brucei spp. have different functions.  One NT carries purine nucleosides (TbNT2) and the other recognizes adenine, adenosine and several antitrypanosomal drugs (TbAT1).  Both of these transporters have a higher affinity for their substrate than do mammalian NTs, which explains why infected patients exhibit a low level of plasma nucleosides (Carter, 2001)

The metacyclic trypanosome NT’s were first discovered in 1980 (James, and Born) and characterized by Carter and Fairlamb in 1993.  Originally the NT’s were named P1 (TbNT2) and P2 (TbAT1), and both had a high affinity for adenosine (fig 5).  TbAT1 was shown to have a Km of 0.59mM and TbNT2 had a Km of 0.15mM.  This study also demonstrated that TbAT1 is inhibited by inosine (fig 6), while TbNT2 is inhibited by adenine (fig 7).  In addition, TbAT1 was shown to be a transporter for phenyl-arsenical drugs such as melarsoprol. This transporter is non-functional in strains that are resistant to melarsoprol. 

In 1998 de Koning, Watson and Jarvis reported on the transport mechanism of the procyclic stage of the parasite, (occurring in the insect vector).  Their research revealed only one type of transporter in this stage of the parasite, similar to TbNT2, that transports adenosine and other purine nucleotides.  This study also revealed a linear correlation between the rate of adenosine transport and proton motive force indicating that it is a proton symporter.  An interesting point here is that the regulation of genes coding for NT in trypanosomes is stage specific, (Sanchez, 1999).  This makes it quite difficult to conquer the parasite with chemotherapeutics; every life cycle stage is a unique mystery.

The first transporter to be molecularly identified was TbAT1, (Maser et al. 1999).   The discovery relied on the yeast, Saccharoyces cerevisiae as an expression system, because they cannot salvage adenosine and normally synthesize it.  The yeast strains used were mutants that were unable to synthesize purines.  The yeast could then be genetically modified with various plasmids from T.b. brucei cDNA libraries, and screened for plasmids that would allow growth in a purine medium.  The survivors were found to have plasmids that coded for a NT, identified as TbAT1.  Determination that this gene coded for the TbAT1, (P2), rather than the TbAT2, (P1), NT was done by testing for the uptake of trypanocidal drugs in yeast expression systems.  The TbAT1 modified yeast were capable of recognizing similar substrates as P2, namely: melaminophenylarsenicals.  Diamidines, however, were not recognized.  Maser suggest that the reason for this is that the yeast may lack some posttranslational modification that is responsible for diamidine recognition. 

The gene was sequenced, and the integral protein was projected to have a structure of ten transmembrane alpha helices and a large intracellular hydrophilic loop between TM 6 and TM 7, (fig 8). Southern blot analysis identified TbAT1 to be a single copy gene.

 The study also looked into the relationship between P2 (TbAT1) and resistance to a variety of antitrypanosomal drugs.  Primarily they proved that defects in TbAT1 did confer resistance to melaminophenylarsenicals.  The resistance may be caused by a mutation that prevents the NT from functioning at all rather than changing substrate specificity, (Maser, 1999).  Recent genetic analysis is bringing rapid assays for resistant strains closer to reality.  Soon it may be possible to use restriction endonucleases and PCR to amplify the aberrant coding regions that have been identified in resistant strains.

            The ability to target resistant strains of Trypanosoma early in infection would greatly aid the fight against sleeping sickness.  The recent developments in membrane topology and gene sequencing of these important nucleotide transporters have advanced current understanding of the therapeutics options available to treat this disease.  Further studies are needed to determine the location of the trypanosome NT’s as well as their driving force.  A better understanding of the actual mechanism by which these NT’s carry their substrates across the membrane against the concentration gradient may lead to new therapies to fight the ever-increasing number of resistant strains.  This type of detailed analysis of nucleotide transporters will apply to many of the parasitic protozoa that utilize similar mechanisms for the uptake of nucleotides from their hosts.

 

 

 

 

 

References

 

Barrett, M.P. and Fairlamb, A.H. (1999) The Biochemical Basis of Arsenical-Diamidine Crossresistance in African Trypanosomes. Parasitology Today 15, 136-140

 

 

Carter, N.S. and Fairlamb, A.H. (1995) Uptake of diamidine drugs by the P2 nucleoside transporter in melarsen resistant Trypanosoma brucei brucei. Journal of Biological Chemistry 270, 28153-28157

 

 

Carter, N.S. Landfear, S.M. and Ullman, B. (2001) Nucleoside transporters in parasitic protozoa. Trends in Parasitology Vol. 17, No.3, 142-145

 

 

De Koning H.P., Watson C.J., Jarvis S.M. (1998) Characterization of a Nucleoside Transporter in Procyclic Trypanosoma brucei brucei.  The Journal of Biological Chemistry Vol. 273, No. 16, 9486-9494.

 

 

De Koning, H.P. and Jarvis, S.M. (2001) Uptake of pentamidine in Trypanosoma brucei brucei is mediated by the P2 adenosine transporter and at least one novel unrelated transporter. Acta Tropic 80, 245-250

 

 

Goldberg B., Rattendi D., Lloyd D., Sufrin J., and Bacchi, C. (2001) In situ kinetic characterization of methyladenosine transport by the adenosine transporter (P2) of the African Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense Biochemical Pharmacology 61, 449-457.

 

 

Harvey, R. et al. (eds) (1997) Lippincotts Illustrated Reviews: Pharmacology, 2^nd ED. Lippincott Williams and Wilkins, 353-356.

 

 

Landfear, Scott M. (2001) Molecular genetics of nucleoside transporters in Leishmania and African Trypanosomes.  Biochemical Pharmacology 62, 149-155

 

 

Maser P., Sutterlin C., Kralli A., and Kaminsky R. (1999) A Nucleoside Transporter  from Trypanosoma brucei Involved in Drug Resistance.  Science 285, 242-244.

Sanchez M.A., Ullman B., Landfear S.M., and Carter N.S. (1999) Cloning and Functional Expression of a Gene Encoding a P1 Type Nucleoside Transporter from Trypanosoma brucei The Journal of Biological Chemistry Vol. 274, No. 42, 30244-30249.

 

 

Suswan E.A., Taylor D.W., Ross C.A., and Martin R.J. (2001) Changes in properties of adenosine transporters in tr5ypanosoma evansi and modes of selection of resistance to the melaminophenyl arsenical drug, Mel Cy. Veterinary Parasitology 102, 193-208