A novel mechanism of mycophenolic acid resistance in the protozoan parasite tritrichomonas foetus

Abstract

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 hypo-xanthine. We have cloned a T.foetus strain (mpa^r) that was 50-fold more resistant to mycophenolic acid than wild type ($\text{ic}{}_{50}\text{= 1}\text{mM}$ for mpa^r vs 20μM for wild type). None of the usual mechanisms of drug resistance could be identified. IMP dehydrogenase isolated from T. foetus mpa^r was indistinguishable from the wild type enzyme. No difference in mycophenolic acid uptake or metabolism was detected between the wild type and mpa^r strains. Mycophenolic acid (100 μM) completely blocked the conversion of adenine and hypoxanthine to guanine nucleotides in T.foetus mpa^r, although no inhibition of T. foetus mpa^r growth was observed at this concentration. These observations indicate that the major purine salvage pathways must be altered in T. foetus mpa^r so that guanine nucleotide biosynthesis no longer requires IMP dehydrogenase. T.foetus mpa^r 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 mpa^r hypoxanthine-guanine-xanthine phosphoribosyltransferases, the key enzyme in purine base incorporation into nucleotides. Two alterations were identified in the purine salvage network of mpa^r: 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 mpa^r. Hypoxanthine and adenine inhibited the incorporation of xanthine into the nucleotide pool in wild type T. foetus, but not in mpa^r. The mpa^r strain, therefore, can salvage xanthine more efficiently from a mixture of purines and thus bypass the drug block at IMP dehydrogenase.