Riboflavin utilisation by the malaria parasite, Plasmodium falciparum, as a target for novel antimalarials
Creators: name: Easton, Kylie Jade
Publisher: name: The Australian National University
Year: 2018
Subjects: subject: QR201.M3 E37 2012
subject: Malaria
subject: Plasmodium falciparum
subject: Vitamin B2

description: The intraerythrocytic malaria parasite Plasmodium falciparum requires an external supply of several essential nutrients, including riboflavin (vitamin B2). In this study, riboflavin-accumulation assays were used to characterise riboflavin transport in the infected erythrocyte and isolated parasite. HPLC analysis confirmed that, once inside the parasite, riboflavin is metabolised into the important coenzymes, FMN and FAD via a similar ATP-dependent pathway as found in other organisms. Consistent with the ability of P. falciparum to metabolise riboflavin in this manner is the identification of two genes encoding putative riboflavin-metabolism enzymes in the parasite's genome. This investigation details heterologous expression and characterisation of these enzymes. The PfFS gene was heterologously expressed in a functionally-active conformation. The protein increased pyrophosphate production in the presence of ATP and FMN, consistent with PfFS having FAD synthetase activity (catalysing the conversion of FMN to FAD). The PfFk gene was not seen to be expressed in soluble form in E. coli and P. pastoris. PfFk was localised as a green fluorescent protein (GFP)-fusion protein to the parasite cytosol, and is not excluded from the nucleus. Parasites expressing the GFP-fusion protein demonstrated increased metabolism of riboflavin to FMN and FAD, consistent with PfFk encoding an enzyme involved in riboflavin metabolism. Riboflavin analogues have previously been shown to inhibit proliferation of P. falciparum. This study demonstrates that the riboflavin analogues roseoflavin and 8-aminoriboflavin possess antiplasmodial activity in the sub-micromolar range and that both analogues significantly inhibit riboflavin metabolism in isolated parasites. It is shown that inside the parasite, roseoflavin is converted to RoFMN and RoFAD. Further investigation into the mechanism of action of riboflavin analogues also showed that a component of the in vitro antiplasmodial activity was due to an effect on host erythrocytes, and that parasite growth in erythrocytes pretreated with either roseoflavin or 8-aminoriboflavin was significantly inhibited. Pretreatment with 8-aminoriboflavin renders uninfected erythrocytes vulnerable to oxidative stress, similar to the effect seen following incubation of erythrocytes in the absence of riboflavin. It has been hypothesised that a familial condition prevalent in Ferrara, Italy, which is characterised by flavin-deficient erythrocytes, provides protection from, and was selected by, malaria. Herein, this hypothesis was investigated via the identification in Ferrara of 35 individuals with flavin-deficient erythrocytes from a total of 150 individuals tested. Using HPLC, it was demonstrated that this deficiency was not diet-related. Erythrocytes from several normal and deficient subjects were then tested for their ability to support P. falciparum growth in vitro. Growth was shown to occur at a significantly lower rate in flavin-deficient erythrocytes, consistent with the condition inhibiting growth of the malaria parasite. This study provides significant advances in knowledge regarding riboflavin uptake and utilisation by P. falciparum. It also demonstrates that riboflavin analogues may provide potent antimalarials with a dual mechanism of action (targeting both the parasite and the host erythrocyte), potentially reducing the parasite's ability to develop drug resistance. The investigations described in this thesis may lead to identification of novel chemotherapies targeting parasite utilisation of riboflavin. -- provided by Candidate.