Targeting Methionine Synthase in a Fungal Pathogen Causes a Metabolic Imbalance That Impacts Cell Energetics, Growth, and Virulence

Abstract

There is an urgent need to develop novel antifungals to tackle the threat fungal pathogens pose to human health. Here, we have performed a comprehensive characterization and validation of the promising target methionine synthase (MetH). We show that in Aspergillus fumigatus the absence of this enzymatic activity triggers a metabolic imbalance that causes a reduction in intracellular ATP, which prevents fungal growth even in the presence of methionine. Interestingly, growth can be recovered in the presence of certain metabolites, which shows that metH is a conditionally essential gene and consequently should be targeted in established infections for a more comprehensive validation. Accordingly, we have validated the use of the tetOFF genetic model in fungal research and improved its performance in vivo to achieve initial validation of targets in models of established infection. We show that repression of metH in growing hyphae halts growth in vitro, which translates into a beneficial effect when targeting established infections using this model in vivo. Finally, a structure-based virtual screening of methionine synthases reveals key differences between the human and fungal structures and unravels features in the fungal enzyme that can guide the design of novel specific inhibitors. Therefore, methionine synthase is a valuable target for the development of new antifungals. IMPORTANCE Fungal pathogens are responsible for millions of life-threatening infections on an annual basis worldwide. The current repertoire of antifungal drugs is very limited and, worryingly, resistance has emerged and already become a serious threat to our capacity to treat fungal diseases. The first step to develop new drugs is often to identify molecular targets in the pathogen whose inhibition during infection can prevent its growth. However, the current models are not suitable to validate targets in established infections. Here, we have characterized the promising antifungal target methionine synthase in great detail, using the prominent fungal pathogen Aspergillus fumigatus as a model. We have uncovered the underlying reason for its essentiality and confirmed its druggability. Furthermore, we have optimized the use of a genetic system to show a beneficial effect of targeting methionine synthase in established infections. Therefore, we believe that antifungal drugs to target methionine synthase should be pursued and additionally, we provide a model that permits gaining information about the validity of antifungal targets in established infections.

Sulfur Metabolism as a Promising Source of New Antifungal Targets

“…In mice, we found that the high doses of doxycycline required to downregulate fungal gene expression in the lungs were toxic to the animals, thus, we could not evaluate the effect on survival. Yet, we could show that the downregulation of metH produced a significant reduction in fungal burden, again similar to the reduction obtained when downregulating cyp51A [ 24 ]. Finally, we performed a structure-based virtual screening demonstrating that fungal methionine synthases have exclusive pockets that can be specifically targeted.…”

“…We firstly investigated the underlying reason for metH essentiality beyond methionine auxotrophy. We found that the absence of methionine synthase triggered a metabolic imbalance, likely due to a forced flow of glucose through the pentose phosphate pathway, that causes a decrease in cell energetics [ 24 ]. Using the tetOFF conditional promoter, we observed that the downregulation of metH in 8 h germinated conidia, as well as in 12 and 16 h-grown mycelia, sustainably halted fungal growth.…”

At the metal–metabolite interface in Aspergillus fumigatus: towards untangling the intersecting roles of zinc and gliotoxin

“…Elevated abundance of zinc-dependent methionine synthase MetH/D was found with addition of 25 µM zinc, and was also found to be induced by heat shock in A. fumigatus [45], is either produced at high zinc due to an activity requirement, or is repressed under zinc-starved conditions. Interestingly there are strong links between the methyl/methionine cycle and GT biosynthesis via the utilization of SAM [46], and the conditional essentiality of MetH has recently been demonstrated [47].…”

Proteomic Response of Paracoccidioides brasiliensis Exposed to the Antifungal 4-Methoxynaphthalene-N-acylhydrazone Reveals Alteration in Metabolism

“…Human pathogenic fungi express all proteinogenic amino acids [ 32 ] and antifungal compounds that inhibit enzymes of amino acid biosynthetic pathways show promising antifungal activity [ 33 , 34 ]. The virtual screening approach showed that inhibiting such enzymes in Aspergillus fumigatus leads to metabolic impairment that causes damage to ATP production, cell growth and virulence [ 34 ]. Here, S-adenosylmethionine synthase is downregulated in P. brasiliensis cells treated with AOS.…”