Rapamycin insensitivity in Schistosoma mansoni is not due to FKBP12 functionality.
Rapamycin (RAPA) is a well-known immunosuppressant, the action of which is mediated by the immunophilin FKBP12. Upon RAPA binding, FKBP12 forms ternary complexes with phosphatidyl inositol related kinases known as the target of RAPA (TOR), which can lead to a mitotic block at the G1-S phase transition. Such an antiproliferative effect makes RAPA an attractive anticancer, antifungal or antiparasitic compound. In this study, we found the helminth parasite Schistosoma mansoni to be insensitive to the drug. In order to elucidate the mechanism underlying RAPA resistance, the S. mansoni drug receptor FKBP12 (SmFKBP12) was cloned for functional analysis. Western blot experiments showed that the protein is constitutively expressed in all life cycle stages and in both male and female parasites. The Escherichia coli-synthesised recombinant protein possessed enzymatic activity, which was inhibitable by RAPA. Moreover, SmFKBP12 was able to complement mutant Saccharomyces cerevisiae cells lacking FKBP12 in their RAPA sensitivity phenotype, leading us to conclude that SmFKBP12 is expressed in yeast in a functional form and capable of interacting with the drug and yeast TOR kinase. Even though the wild type SmFKBP12 appeared to restore a large part of RAPA sensitivity, a mutation of Asp(89)-Lys(90) to Pro(89)-Gly(90) in the schistosome protein was found to be more effective and restored drug sensitivity to the same level as the endogenous yeast protein. Despite ternary complex formation, our results suggest that additional unknown factors other than a functional drug receptor are implicated in drug resistance mechanisms.
Rossi
Alessandro
A
Department of Parasitology, Institute for Tropical Medicine, University of Tübingen, Wilhelmstrasse 27, 72074 Tubingen, Germany.
Pica-Mattoccia
Livia
L
Cioli
Donato
D
Klinkert
Mo-Quen
MQ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012550:Schistosoma mansoni / Q000187:drug effects* / Q000378:metabolism; D012556:Schistosomicides / Q000378:metabolism / Q000494:pharmacology*; D016415:Sequence Alignment; D020123:Sirolimus / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology*; D022061:Tacrolimus Binding Protein 1A / Q000235:genetics / Q000502:physiology*; D015003:Yeasts / Q000378:metabolism
D007166:Immunosuppressive Agents; D011994:Recombinant Proteins; D012556:Schistosomicides; D022061:Tacrolimus Binding Protein 1A; D019696:Peptidylprolyl Isomerase; D020123:Sirolimus