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

Abstract

29There is an urgent need to develop novel antifungals to tackle the threat fungal pathogens pose 30 to human health. In this work, we have performed a comprehensive characterisation and 31 validation of the promising target methionine synthase (MetH). We uncover that in Aspergillus 32 fumigatus the absence of this enzymatic activity triggers a metabolic imbalance that causes a 33 reduction in intracellular ATP, which prevents fungal growth even in the presence of methionine. 34Interestingly, growth can be recovered in the presence of certain metabolites, which evidences 35 that conditional essentiality, defined as genes whose deficiency can be overcome in specific 36 conditions, is present in pathogenic fungi. As this concept must be considered for correct target 37 validation, we have optimised a genetic model to mimic treatment of established infections 38 using the tetOFF system. We show that repression of metH in growing hyphae halts growth in 39 vitro, which translates into a beneficial effect when targeting established infections using this 40 model in vivo. Finally, a structural-based virtual screening of methionine synthases reveals key 41 differences between the human and fungal structures and unravels features in the fungal 42 enzyme that can guide the design of novel specific inhibitors. Therefore, methionine synthase is 43 a valuable target for the development of new antifungals. 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59Fungal pathogens represent an increasing risk to human health 1 , with over one billion people 60 worldwide affected by mycoses annually. Many of these mycoses are superficial infections of 61 the skin, nails or mucosal membranes and although troublesome are usually not life-62 threatening. However, some fungi cause devastating chronic and invasive fungal infections, 63 which result in an estimated 1.6 million deaths per year 2 . Incidences of invasive infections 64 caused by Aspergillus, Candida, Cryptococcus and Pneumocystis species are increasing 3 , a cause 65 for serious concern as these genera are responsible for 90% of deaths caused by mycoses 4 . 66Despite the availability of antifungal drugs, mortality rates for invasive aspergillosis, invasive 67 candidiasis, cryptococcal meningitis and Pneumocystis jirovecii pneumonia are intolerably high, 68reaching over 80%, 40%, 50% and 30% respectively 2,5 . There are currently only four classes of 69 antifungals in clinical use to treat invasive infections (azoles, echinocandins, polyenes and 70 flucytosine), all suffering from pharmacological drawbacks including toxicity, drug-drug 71 interactions and poor bioavailability 6,7 . With the sole exemption of flucytosine, which is only 72 used in combinatory therapy with amphotericin B for cryptococcal meningitis and Candida 73 endocarditis 7 , the current antifungals target critical components of the fungal cell membrane 74 or cell wall 8 , which represents a very limited druggable space. The rise of antifungal resistance 75 presents an additional challenge as mortality rates in pat...