Host Genetics and Responses - Project D3
The antiplasmodial activity of riboflavin analogues
Host lab: Kevin Saliba (ANU)
Partner lab: Frank Seeber (RKI)
´Background: Riboflavin (vitamin B2) is converted into flavin mononucleotide (FMN) by riboflavin kinase, and subsequently metabolised into flavin adenine-dinucleotide (FAD) by FAD synthetase. FMN and FAD, referred to as flavins, serve as cofactors for a number of essential enzymes. Several riboflavin analogues have been shown to possess antiplasmodial activity during the intraerythrocytic stage of the malaria parasite. We have been focusing on the antiplasmodial activity of two specific riboflavin analogues, roseoflavin and 8-amino-riboflavin. Roseoflavin has been shown to be metabolised into antimetabolites of FMN and FAD, whereas 8-amino-riboflavin appears to only be converted into an FMN antimetabolite.
We have generated roseoflavin resistant parasites by exposing intraerythrocytic parasites to sub-lethal concentrations of roseoflavin over a number of weeks. Whole genome sequencing of the resistant parasites identified a single point mutation in riboflavin kinase. These parasites are cross-resistant to 8-amino-riboflavin. Preliminary data indicate that T. gondii parasites are sensitive to riboflavin analogues.
Hypotheses: (i) That riboflavin antimetabolites kill the parasite by inhibiting FMN and FAD-dependent enzymes. (ii) That mutations in riboflavin metabolising enzymes confer resistance to riboflavin analogues by modifying the affinity of the enzymes to the analogues.
1) Generate P. falciparum parasites that are resistant to 8-amino-riboflavin, followed by whole genome sequencing. (ANU)
2) Generate transgenic parasites (T. gondii and P. falciparum) overexpressing either FMN or FAD-dependent enzymes (candidates to be determined at a later stage). It is expected that over-expression of FAD-dependent proteins will have no effect on 8-amino-riboflavin sensitivity, but overexpression of FMN-dependent proteins will convey resistance. Overexpression of either FMN or FAD-dependent proteins is expected to result in roseoflavin resistance. (ANU, P. falciparum work, RKI, T. gondii work)
3) Characterise the riboflavin kinase mutation in order to understand how it conveys resistance to the riboflavin analogues. The mutated residue appears to be conserved across different organisms. A riboflavin kinase from a suitable organism that has been previously expressed heterologously can be mutated, expressed, purified and the enzyme activity characterised (including its ability to metabolise the riboflavin analogues). (ANU)
4) Introduce the riboflavin resistance-conferring mutations observed in P. falciparum into the equivalent genes in T. gondii and determine whether they also confer resistance to the analogues in T. gondii. (RKI)
Keywords: Riboflavin, Antimetabolites, Mechanism of Action, Protein expression, Whole Genome Sequencing, Enzyme Assays.
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