Leishmania infantum parasites resistant to the S-adenosylmethionine Analogue Sinefungin

To further our understanding of one carbon metabolism in the protozoan parasite Leishmania we carried genomic screens to study how the parasite responded to sinefungin (SNF) selection. SNF is a structural analogue of S-adenosylmethionine (AdoMet), a key methyl group donor to a number of biomolecules. One screen consisted in sequencing SNF resistant mutants generated by step-wise selection in gradually increasing drug concentrations. These studies demonstrated deletion of the AdoMet transporter (AdoMet1) by intergenic recombination as a crucial loss of function marker for SNF-resistance. The second screen consisted in Cos-seq, a gain-of-function cosmid based genome wide functional screen with increasing SNF concentration coupled to next generation sequencing. Cosmids enriched in that screen and sequenced led to the identification of i) the AdoMet-synthetase (METK) as the major SNF-target; ii) a mRNA (guanine-N(7)-)-methyltransferase (CMT1); iii) a leucine carboxyl methyltransferase (LCMT); iv) two tryparedoxin genes and v) two protein phosphatase regulatory genes. Further functional exploration indicated that LCMT interacts with one phosphatase catalytic subunit (PP2AC) and mutation of the C-terminal leucine residue of PP2AC affects sinefungin susceptibility. These holistic screens led to the identification of transporters, biosynthetic genes, RNA and protein methyltransfereases as well as phosphatases linked to AdoMet mediated functions in Leishmania.