Mutation of the P. falciparum flavokinase confers resistance to roseoflavin and 8-aminoriboflavin

Abstract

We previously found that two riboflavin analogues, roseoflavin and 8-aminoriboflavin, inhibit malaria parasite proliferation by targeting riboflavin utilisation. To determine the mechanism of action of roseoflavin in P. falciparum, we generated roseoflavin-resistant parasites by in vitro evolution over 27 weeks. The roseoflavin-resistant parasites were found to be four times more resistant to roseoflavin and cross-resistant to 8-aminoriboflavin. Resistant parasites were subjected to whole genome sequencing and a missense mutation (T2015A), leading to an amino acid exchange (L672H), was detected in the gene coding for a putative flavokinase (PfFK), the enzyme responsible for converting riboflavin (vitamin B2) into the cofactor flavin mononucleotide (FMN). To confirm that the L672H mutation is responsible for the observed phenotype, we generated parasites with the missense mutation incorporated into the PfFK gene via a single-crossover recombination. The IC50 values for roseoflavin (RoF) and 8-aminoriboflavin against the RoF-resistant parasites created through in vitro evolution were indistinguishable from the IC50 values for parasites in which the missense mutation was specifically introduced into the native PfFK. To investigate this mutation, we generated two parasite lines episomally-expressing GFP-tagged versions of either the wild type or mutant forms of flavokinase. We found that PfFK-GFP localises to the parasite cytosol and that immunopurified PfFK-GFP was active and phosphorylated riboflavin into flavin mononucleotide. The L672H mutation caused a reduction of the binding affinity, especially for the substrate RoF, which explains the resistance phenotype. The mutant PfFK is no longer capable of phosphorylating 8-aminoriboflavin, but its antiplasmodial activity against resistant parasites can still be antagonised by increasing the extracellular concentration of riboflavin, consistent with the compound also inhibiting parasite growth through competitive inhibition of PfFK. Our findings, therefore are consistent with roseoflavin and 8-aminoriboflavin inhibiting parasite growth by inhibiting FMN production, in addition to the generation of toxic flavin cofactor analogues.