A novel mechanism of mycophenolic acid resistance in the protozoan parasite Tritrichomonas foetus.
Tritrichomonas foetus relies primarily on the salvage of hypoxanthine to supply purine nucleotides. Mycophenolic acid disrupts T. foetus growth by specifically inhibiting inosine-5'-monophosphate (IMP) dehydrogenase, thereby blocking the biosynthesis of guanine nucleotides from hypoxanthine. We have cloned a T. foetus strain (mpar) that was 50-fold more resistant to mycophenolic acid than wild type (IC50 = 1 mM for mpar vs 20 microM for wild type). None of the usual mechanisms of drug resistance could be identified. IMP dehydrogenase isolated from T. foetus mpar was indistinguishable from the wild type enzyme. No difference in mycophenolic acid uptake or metabolism was detected between the wild type and mpar strains. Mycophenolic acid (100 microM) completely blocked the conversion of adenine and hypoxanthine to guanine nucleotides in T. foetus mpar, although no inhibition of T. foetus mpar growth was observed at this concentration. These observations indicate that the major purine salvage pathways must be altered in T. foetus mpar so that guanine nucleotide biosynthesis no longer requires IMP dehydrogenase. T. foetus mpar incorporated xanthine more efficiently into the nucleotide pool relative to hypoxanthine and guanine than wild type. Xanthine incorporation via XMP provided an IMP dehydrogenase independent route to guanine nucleotides that would enable the parasite to become mycophenolic acid resistant. No difference could be detected between wild type and mpar hypoxanthine-guanine-xanthine phosphoribosyltransferases, the key enzyme in purine base incorporation into nucleotides. Two alterations were identified in the purine salvage network of mpar: it was deficient in hypoxanthine transport and had diminished adenine deaminase activity. The apparent net result of these two changes was to lower the intracellular concentration of hypoxanthine in mpar. Hypoxanthine and adenine inhibited the incorporation of xanthine into the nucleotide pool in wild type T. foetus, but not in mpar. The mpar strain, therefore, can salvage xanthine more efficiently from a mixture of purines and thus bypass the drug block at IMP dehydrogenase.
Hedstrom
L
L
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143.
Cheung
K S
KS
Wang
C C
CC
D000225:Adenine / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular; D004351:Drug Resistance / Q000235:genetics; D006147:Guanine / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism; D019271:Hypoxanthine; D007041:Hypoxanthine Phosphoribosyltransferase / Q000378:metabolism; D007042:Hypoxanthines / Q000378:metabolism; D007168:IMP Dehydrogenase / Q000037:antagonists & inhibitors*; D007658:Ketone Oxidoreductases / Q000037:antagonists & inhibitors*; D009154:Mutation; D009173:Mycophenolic Acid / Q000378:metabolism / Q000494:pharmacology*; D014318:Tritrichomonas / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D019820:Xanthine; D014970:Xanthines / Q000378:metabolism
D006150:Guanine Nucleotides; D007042:Hypoxanthines; D014970:Xanthines; D019820:Xanthine; D019271:Hypoxanthine; D006147:Guanine; D007168:IMP Dehydrogenase; D007658:Ketone Oxidoreductases; D007041:Hypoxanthine Phosphoribosyltransferase; D009173:Mycophenolic Acid; D000225:Adenine