Purpose: We have previously demonstrated that the methionine synthase encoding gene (metH) is essential for Aspergillus fumigatus viability and virulence, which led us to propose it as candidate for antifungal drug development. The mechanistic basis of its essentiality is unknown, but much-needed in order to fully explore the therapeutic potential of this enzyme as a drug target. Therefore, our aim is to unravel the underlying reason for methionine synthase essentiality in order to verify its suitability for drug development.
Methods: We have constructed a strain which expresses the metH gene under the control of the regulatable tetOFF system. We have tried to rescue growth of this strain under restrictive conditions following a number of strategies designed to discover the mechanistic basis of essentiality.
Results: Previously we did not determine what is essential, integrity of the methionine biosynthesis route or the methionine synthase protein per se. To discern between these two possibilities, we attempted to construct a double metGΔcysDΔ mutant, which is blocked in the previous step of the pathway. This strain turned out to be a viable methionine auxotroph, demonstrating that it is the methionine synthase enzyme that is crucial for viability. Next we aimed to find out whether its essentiality is dependent or not on its catalytic action. Lack of enzymatic activity could result in accumulation of the potentially toxic substrate homocysteine and/or in shortage of the by-product tetrahydrofolate (THF), which is required for nucleotide biosynthesis. To determine if the catalytic action is required, we have first tried to rescue growth of the metH_tetOFF strain by ameliorating those potential negative effects. We have episomally overexpressed the cystathionine-beta-synthase encoding gene using a newly designed AMA1 self-replicating plasmid. Cystathionine-beta-synthase uses homocysteine as substrate and therefore its overexpression should prevent toxic accumulation. We have also supplemented the media with THF and/or nucleotides, to circumvent growth blockade caused by their deficiency. However, growth was not rescued in single or combinatorial experiments, suggesting that lack of enzymatic activity per se cannot explain methionine synthase essentiality. In the next experiment, we have expressed two different inactive versions of the enzyme (point-mutated in the catalytic and substrate binding sites, respectively) in the metH_tetOFF strain. Under restrictive conditions, both strains are viable methionine auxotrophs, proving that essentiality is indeed independent of methionine biosynthetic activity. We are currently performing metabolome analyses to determine if the absence of the protein, but not its normal enzymatic activity, triggers a deleterious re-programming of the metabolism which could explain its essentiality and shed light on the underlying mechanism.
Conclusion: Methionine synthase is essential in Aspergillus fumigatus, but interestingly this is independent of its enzymatic action, which suggests that the protein have additional, still unidentified, crucial roles.
Full conference title:
- AAA 8th (2018)