Mechanism of resistance of Pediococcus cerevisiae strains to 5-fluorodeoxyuridine

Abstract

The Pediococcus cerevisiae strains, the wild-type (ATCC 8081), a folate-permeable strain, PteGlu, and a folate-permeable amethopterin-resistant strain, AMr, were made resistant to 5-fluoro-2′-deoxyuridine (FdUrd) by successive transfer in increasing concentrations of the drug. The selected mutant strains, wild-type FdUrd, PteGluFdUrd and AMeFdUrd exhibited a 10 7-fold greater tolerance to FdUrd than the parents. The activities of folate-related enzymes did not change with the development of resistance. Dihydrofolate reductase (5, 6,7,8-tetrahydrofolate: NADP + oxidorductase, EC 1.5.1.3) was of almost the same activity in the FdUrd-susceptible parents as in the resistant mutants. Thymidylate synthetase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) activity and its susceptibility to inhibition by 5-fluoro- 2′-deoxyuridine-5′-monophosphate (FdUMP) were the same in the parents as in the mutants. Two factors were found to be responsible for the resistance to FdUrd: (1) a decrease or lack of activity of thymidine kinase (ATP; thymidine- 5′-phosphotransferase, EC 2.7.1.21) and (2) an impaired cell permeability to FdUrd. After incubation for 1 min the susceptibility strains accumulated radioactive FdUrd up to 45-fold (wild-type) and 90-fold (PteGlu and AMr strains) higher intracellular concentration than that of the medium. About 50% of the accumulated radioactivity was FdUMP. The wild-type FdUrd mutant could not accumulate the drug against the concetration gradient, whereas the PteGlu FdUrd and AMr FdUrd strains accumulated the substrate up to an 8 times higher intracellular concentration than in the medium. The radioactivity identified in the medium after incubation with the susceptibility and resistant cells was intact FdUrd. Thus, no degradation of FdUrd to fluorouracil (FUra) occured prior to uptake, nor was FUra released from the cells into the medium. The uptake of FdUrd in the parents and in the mutants was shown to be glucose-dependent and inhibited by deoxyuridine and thymidine.