Sueiro-Olivares, M. | bioRxiv Science

bioRxiv2024

The sulfur-related metabolic status of Aspergillus fumigatus during infection reveals cytosolic serine hydroxymethyltransferase as a promising antifungal target.

Sulfur metabolism is an essential aspect of fungal physiology and is known to be crucial for pathogenicity. Fungal sulfur metabolism comprises anabolic and catabolic routes that are not well- conserved in mammals, and therefore can be considered a promising source of prospective novel antifungal targets. To gain insight into the status of the Aspergillus fumigatus sulfur-related metabolism during infection we used a NanoString custom nCounter TagSet and compared the expression of 68 key metabolic genes in different murine models of invasive pulmonary aspergillosis, at three different time-points, and a variety of in vitro conditions. We identified a set of 15 genes that are consistently expressed at higher levels in vivo than in vitro, suggesting that they may be particularly relevant for intrapulmonary growth and therefore constitute promising drug targets. Indeed, the role of five of the fifteen genes had previously been empirically validated, supporting the likelihood that the remaining candidates are relevant. In addition, the analysis of the dynamics of gene expression at the early (16h), mid (24h-1) and late (72h) time-points uncovered potential disease initiation and progression factors. We further characterised one of the identified genes, encoding the cytosolic serine hydroxymethyltransferase ShmB, and demonstrated that it is an essential gene of A. fumigatus and that it is also required for virulence in a murine model of established pulmonary infection. We further show that the structure of the ligand binding pocket of the fungal enzyme differs significantly from its human counterpart, suggesting that specific inhibitors can be designed. Therefore, in vivo transcriptomics is a powerful tool to identify genes crucial for fungal pathogenicity that might encode promising antifungal target candidates. AUTHOR SUMMARYAspergillus fumigatus is an opportunistic human fungal pathogen that causes devastating chronic and invasive infections in immunocompromised patients. Our arsenal of antifungal drugs to fight this and other fungal pathogens is very limited, partly because of the high similarity between eukaryotic fungal and human cells makes the identification of suitable drug targets a challenging task. Furthermore, targets identified in vitro are often not effective in vivo, as their action is not relevant for fungal virulence. To address this challenge, we compared the expression profiles of a set of genes involved in sulfur metabolism, a promising source of potential drug targets, in numerous in vitro and in vivo conditions to identify favourable antifungal candidates. Subsequently, we validated one of the highlighted genes, demonstrating that it is essential for A. fumigatus viability and virulence, and that it can likely be targeted by specific inhibitors. Hence, we show the potential of using in vivo transcriptomics to identify targets that contribute to virulence, propose various candidates for future studies and present a novel target validated for further antifungal drug development.

microbiology↗

bioRxiv2020

Targeting methionine synthase in a fungal pathogen causes a metabolic imbalance that impacts cell energetics, growth and virulence

There is an urgent need to develop novel antifungals to tackle the threat fungal pathogens pose to human health. In this work, we have performed a comprehensive characterisation and validation of the promising target methionine synthase (MetH). We uncover 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 evidences that metH is a conditionally essential gene. As this implies that for a correct validation MetH should be targeted in established infections, we have validated the use of the tetOFF genetic model for fungal research and optimised its performance to mimic treatment of established infections. 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 structural-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. IMPORTANCEFungal 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 often is to identify molecular targets which inhibition during infection can prevent pathogen growth. However, the current models are not suitable to validate targets in established infections. Here we have characterised 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 optimised 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 propose that antifungal targets should be validated in a model of established infection.

microbiology↗