ABSTRACT
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 conditional essentiality, defined as genes whose deficiency can be overcome in specific conditions, is present in pathogenic fungi. As this concept must be considered for correct target validation, we have optimised a genetic model to mimic treatment of established infections using the tetOFF system. 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.
INTRODUCTION
Fungal pathogens represent an increasing risk to human health 1, with over one billion people worldwide affected by mycoses annually. Many of these mycoses are superficial infections of the skin, nails or mucosal membranes and although troublesome are usually not life-threatening. However, some fungi cause devastating chronic and invasive fungal infections, which result in an estimated 1.6 million deaths per year 2. Incidences of invasive infections caused by Aspergillus, Candida, Cryptococcus and Pneumocystis species are increasing 3, a cause for serious concern as these genera are responsible for 90% of deaths caused by mycoses 4. Despite the availability of antifungal drugs, mortality rates for invasive aspergillosis, invasive candidiasis, cryptococcal meningitis and Pneumocystis jirovecii pneumonia are intolerably high, reaching over 80%, 40%, 50% and 30% respectively 2,5. There are currently only four classes of antifungals in clinical use to treat invasive infections (azoles, echinocandins, polyenes and flucytosine), all suffering from pharmacological drawbacks including toxicity, drug-drug interactions and poor bioavailability 6,7. With the sole exemption of flucytosine, which is only used in combinatory therapy with amphotericin B for cryptococcal meningitis and Candida endocarditis 7, the current antifungals target critical components of the fungal cell membrane or cell wall 8, which represents a very limited druggable space. The rise of antifungal resistance presents an additional challenge as mortality rates in patients with resistant isolates can reach 100%, making the development of new antifungal drugs increasingly critical for human health 1,9. Targeting fungal primary metabolism is broadly considered a valid strategy for the development of novel antifungals, as it is crucial for pathogen virulence and survival 10,11. A primary example of success of this strategy is olorofim (F901318), a novel class of antifungal that targets the pyrimidine biosynthesis pathway 12, which is currently in clinical trials.
Methionine synthases catalyse the transfer of a methyl group from N5-methyl-5,6,7,8-tetrahydrofolate (CH3-THF) to L-homocysteine (Hcy). Two unrelated protein families catalyse this reaction: cobalamin dependent methionine synthases (EC 2.1.1.13) and cobalamin independent methionine synthases (EC 2.1.1.14). Members of both families must catalyse the transfer of a low active methyl group from the tertiary amine, CH3-THF, to a relatively weak nucleophile, Hcy sulfur. Cobalamin dependent enzymes facilitate this transfer by using cobalamin as an intermediate methyl carrier 13. By contrast, cobalamin independent enzymes directly transfer the methyl group from CH3-THF to Hcy 14. Logically, proteins of each family differ significantly both at amino acid sequence 15 and 3D structure level 16.
We have previously shown that the methionine synthase-encoding gene is essential for A. fumigatus viability and virulence, which led us to propose it as a promising target for antifungal drug development 17. In support of this, a systematic metabolic network analysis by Kaltdorf and colleagues identified methionine synthase as a promising antifungal drug target worthy of investigation 18. Methionine synthase has also been described as essential for Candida albicans viability 19,20 and necessary for Cryptococcus neoformans pathogenicity 21, which suggests that a drug developed against this enzyme may have a broad spectrum of action. Moreover, fungal methionine synthases are cobalamin independent, differing significantly from the cobalamin dependent human protein at the amino acid sequence level: only 11.2% identity, 20.4% similarity and 60.2% gaps when aligned the A. fumigatus and human proteins using L-Align from EMBL 22,23. Therefore, it should be possible to develop drugs with low toxicity potential.
Target validation is critical and has been suggested as the most important step in translating a new potential target into a viable drug target because of its role in achieving efficacy in patients 24. Indeed a retrospective analysis from AstraZeneca’s drug pipeline showed that projects that had performed a more thorough target validation were less likely to fail: 73% of the projects were active or successful in Phase II compared with only 43% of projects without such extra target validation 25. Therefore, in this work we aimed to further substantiate methionine synthase’s potential as an antifungal drug target, before advancing the drug discovery process. In particular, we were interested in 1) unravelling the mechanistic basis of methionine synthase essentiality in A. fumigatus, which is needed to fully explore the potential of this enzyme as drug target and to be able to anticipate drug resistance mechanisms; and 2) developing in vivo models of infection to mimic treatment against the target in an established infection and using them to validate methionine synthase as an antifungal drug target.
RESULTS AND DISCUSSION
Methionine synthase enzymatic activity is essential for Aspergillus fumigatus viability
We had previously demonstrated that the methionine synthase encoding gene is essential for A. fumigatus viability and virulence 17; however, the underlying reason for this essentiality was still unclear. To address this question, here we have constructed strains that express the metH gene under the control of a tetOFF system recently adapted for Aspergillus 26 in two different A. fumigatus wild-type backgrounds, ATCC46645 and A1160. The advantage of the tetOFF system over other regulatable systems is that doxycycline (Dox) can be added to downregulate gene expression in growing hyphae (Fig. S1A), and thus this system permits investigation of the consequences of the repression of an essential gene in growing mycelia. The constructed metH_tetOFF strains (H_OFF) grew as the wild type in the absence of Dox, but as little as 0.5 µg/mL was sufficient to completely prevent colony development on an agar plate even in the presence of methionine (Fig. S1B). This corroborates our previous result that methionine synthase is essential for A. fumigatus viability and that its absence does not result in a sheer auxotrophy for methionine. 17.
Methionine synthase forms an interjection between the trans-sulfuration pathway and the one carbon metabolic route (Fig. 1A), as the enzyme utilizes 5-methyl-tetrahydrofolate as co-substrate. Therefore, the essentiality of metH might be due to required integrities of the trans-sulfuration pathway or of the one carbon metabolic route. Alternatively, it could be that the presence of the enzyme itself is essential, either because its enzymatic activity is required or because it is fulfilling an unrelated additional role, as being part of a multiprotein complex. To start discerning among these possibilities, we constructed a double ΔmetGΔcysD mutant, blocked in the previous step of the trans-sulfuration pathway, and a ΔmetF deletant, which blocks the previous step of the one-carbon metabolic route (Fig. 1A). As we had previously observed 27, to rescue fully ΔmetF’s growth the media had to be supplemented with methionine and other amino acids, as the folate cycle is necessary for the interconversion of serine and glycine and plays a role in histidine and aromatic amino acid metabolism 28,29. Consequently, we added a mix of all amino acids except cysteine and methionine to the S-free medium for this experiment. Phenotypic tests (Fig. 1B) confirmed that the ΔmetGΔcysD and ΔmetF mutants were viable and could grow in the presence of methionine. In contrast, the H_OFF conditional strain could not grow under restrictive conditions even in the presence of the amino acid mix and methionine (Fig. 1B). Therefore, the MetH protein itself, and not the integrity of the trans-sulfuration and one-carbon pathways, is essential for A. fumigatus viability. Interestingly, the methionine auxotroph ΔmetGΔcysD was avirulent in a leukopenic model of pulmonary aspergillosis (Fig. S1C), suggesting that the amount of readily available methionine in the lung is very limited, not sufficient to rescue its auxotrophy. Indeed, the level of methionine in human serum was calculated to be as low as ∼20 µM 30,31, which was described as insufficient to support the growth of various auxotrophic bacterial pathogens 32 and we have also observed that is not enough to rescue growth of the A. fumigatus ΔmetGΔcysD auxotroph.
Essentiality of the MetH protein could be directly linked to its enzymatic activity or, alternatively, the protein could be performing an additional independent function. To discern between these two possibilities, we constructed two strains that express single-point mutated versions of MetH from the innocuous Ku70 locus of the H_OFF background strain, under the control of its native promoter (Fig. 1C). These point mutations, metHg2042A>2C (D616A) and metHg2179TA>9GC (Y662A), were previously described to prevent conformational rearrangements required for activity of the C. albicans methionine synthase 33. In the absence of Dox, these strains grew normally, as they expressed both the wild-type MetH, from the tetOFF promoter, and the mutated version of the protein (Fig. 1C). In the presence of Dox, when the wild-type metH gene was downregulated, the Y662A strain grew on sulfate worse than in non-restrictive conditions, but still to a significant extent, suggesting that this point mutation did not completely abrogate enzymatic activity (Fig. 1C). Interestingly, Y662 grew normally on methionine, showing that methionine can compensate for a partial reduction of MetH activity (Fig 1C). The D616A strain (two isolates were tested) was not able to grow on sulfate (Fig. 1C), demonstrating that enzymatic activity was fully blocked. Nor could it grow on methionine (Fig. 1C), indicating that enzymatic activity is required for viability even in the presence of the full protein. All these phenotypes support the conclusion that methionine synthase enzymatic activity is required for viability.
Absence of methionine synthase enzymatic activity results in a shortage of crucial metabolites, but does not cause toxic accumulation of homocysteine
The absence of methionine synthase enzymatic activity has two direct consequences, which could cause deleterious effects and therefore explain its essentiality (Fig. 1A). It could cause an accumulation of the potentially toxic substrate homocysteine and a shortage of the co-product tetrahydrofolate (THF). THF is directly converted to 5,10-methylene-THF, which is required for the synthesis of purines and thymidylate (TMP), and thus for DNA synthesis; additionally, as purine biosynthesis requires Gln, Gly and Asp, and THF de novo synthesis requires chorismate (precursor of aromatic amino acids), a shortage of THF might cause a depletion of amino acids (Fig. 1A). To investigate if the depletion of any of these metabolites underlies MetH essentiality, we supplemented the media with a number of precursors and potentially depleted metabolites (Fig. 2A). Added as sole supplement, only adenine was able to trigger growth, but to a minimal degree. Further addition of a mixture of all amino acids noticeably improved growth. Supplementation with adenine and guanine (purine bases) did also reconstitute noticeable growth, which was not enhanced with further addition of amino acids. Folic acid was also capable of reconstituting growth, but only when amino acids were added as the sole N-source (Fig. S3). However, no combination of compounds was able to reconstitute growth to the wild-type level. This suggests that a shortage of relevant metabolites derived from THF, prominently adenine, partially accounts for methionine synthase essentiality, but cannot explain it completely. Hence, although supplementation with adenine and other amino acids can partially restore growth, a metH mutant in A. fumigatus is not merely a methionine and adenine combined auxotroph, as it is the case in other fungi as Pichia pastoris 34 or Schizosaccharomyces pombe 35.
To investigate if homocysteine could be accumulating to toxic levels in the absence of MetH activity, we over-expressed several genes that should alleviate its accumulation. To this aim we designed and constructed the plasmid pJA49, which allows direct integration of any ORF to episomally overexpress genes in A. fumigatus. Plasmid pJA49 carries the A. nidulans AMA1 autologous replicating sequence 36,37 and the hygromycin B resistance gene (hygrB) as a selection marker. A unique StuI restriction site allows introduction of any PCR amplified ORF in frame under the control of the A. fumigatus strong promoter hspA 38 and the A. nidulans trpC terminator (Fig S2A). Using this plasmid, we produced a strain in the H_OFF background that episomally overexpresses mecA, encoding cystathionine-β-synthase, which converts homocysteine to cystathionine (Fig. 1A). Homocysteine exerts toxic effects through its conversion to S-adenosylhomocysteine, which causes DNA hypomethylation 39,40, or to homocysteine thiolactone, which causes N-homocysteinylation at the ε-amino group of protein lysine residues 41,42. Consequently, we also constructed strains that episomally over-express genes that could detoxify those products: the S-adenosyl-homocysteinase lyase encoding gene sahL (AFUA_1G10130) or the A. nidulans homocysteine thiolactone hydrolase encoding gene blhA (AN6399) (A. fumigatus genome does not encode any orthologue) (Fig. S2B). However, despite a strong over-expression of the genes (Fig. S2C&D), none of them could rescue growth of the H_OFF strain in restrictive conditions (Fig. 2B), suggesting that a toxic accumulation of homocysteine does not explain the essentiality of MetH enzymatic activity. Addition of adenine to the medium did not improve growth of the overexpression strains further than that of the H_OFF background (Fig. 2B), indicating that there is not a combined effect of homocysteine accumulation and depletion of THF-derived metabolites. Toxic accumulation of homocysteine was speculated to be the underlying reason of methionine synthase essentiality in both Candida albicans and Cryptococcus neoformans 20,21 but we have demonstrated that this is not the case in A. fumigatus. Therefore, although we do not have any evidence to refute the previous assumption in other fungal pathogens, we suggest that it should be revisited.
Methionine synthase repression triggers a metabolic imbalance that causes a decrease in cell energetics
Aiming to identify any adverse metabolic shift in the absence of MetH and/or accumulation of toxic compounds that could explain its necessity for proper growth, we performed a metabolomics analysis, via gas chromatography-mass spectrometry (GC-MS), comparing the metabolites present in wild-type and H_OFF strains before and after Dox addition. Before Dox addition both strains clustered closely together in a Principal Component Analysis (PCA) scores plot (Fig. S4A), showing that their metabolic profiles are highly similar. However, 6 h after Dox addition the strains clusters became clearly separated, denoting differential metabolite content. Analysis of the differentially accumulated metabolites (full list can be consulted in Table S1) using the online platforms MBRole 43 and Metaboanalyst 44,45 did not reveal any obvious metabolic switch, probably due to the rather small number of metabolites that could be identified by cross-referencing with the Golm library (http://gmd.mpimp-golm.mpg.de/). Manual inspection of the metabolites pointed out interesting aspects. Firstly, the methionine levels were not significantly different, which demonstrates that methionine supplementation in the growth medium triggers correct intracellular levels in the H_OFF strain; this undoubtedly rules out that a shortage of methionine could be the cause of the essentiality of methionine synthase. Secondly, we detected a significantly lower amount of adenosine in the H_OFF strain compared with the wild-type after Dox addition (Fig. 3A), which is in agreement with our previous result that supplementation of adenine can partially reconstitute growth in the absence of MetH. We did not find accumulation of compounds with a clear toxic potential upon metH repression. Nevertheless, we detected a lower amount of several amino acids (Phe, Ser, Glu, Pro, Ile, Thr, Ala and Asp, Fig. S4B), which suggests that the cells may enter into growth arrest upon metH repression. Interestingly, we noticed a significantly lower accumulation of some metabolites of the glycolysis pathway and TCA cycle (Fig. 3A) and some other mono and poly-saccharides (Fig. S4B). These variations could reflect a low energetic status of the cells upon metH repression. Indeed, we found that the level of ATP significantly decreased in the H_OFF strain upon Dox addition (Fig. 3B). Therefore, we evaluated if supplementation of the medium with substrates that have the potential to increase cell energetics can rescue H_OFF growth in restrictive conditions. Indeed, we found that when pyruvate, which can directly be converted to acetyl-CoA to enter the TCA cycle, was added as the sole carbon source H_OFF growth was reconstituted in restrictive conditions to the same level as the wild-type (Fig. 3C). Growth was limited for both strains, as pyruvate does not appear to be a good carbon source 46. However, the presence of glucose in the medium precluded the reconstitution of growth of H_OFF (Fig. S3), as it has been described to prevent pyruvate uptake in S. cerevisiae 47. We next tested the capacity of ATP to be used an alternative energy source and to reconstitute growth. To diversify the presence of permeases in the cell membrane, and thus maximise the chance of ATP uptake, we assayed two different N-sources: ammonium (NH4+, preferred source) and amino acids (Fig. S3). Indeed, when amino acids were the only N-source, supplementation of the medium with ATP reconstituted H_OFF growth in restrictive conditions to wild type levels (Fig. 3C). This agrees with the recent observation that eukaryotic cells can uptake ATP and exploit it as an energy source 48. In conclusion, a decrease in cell energetics developed in the absence of methionine synthase is the underlying reason of its essentiality for growth.
The fact that growth in the absence of methionine synthase can be reconstituted when there are sufficient levels of ATP implies that metH is not an absolutely essential gene. It falls within the definition of conditional essentiality, which encompasses genes that are essential in the absence of the specific conditions that can overcome disturbances derived from its deficiency. We expect that a significant number of genes previously described as essential in fungi would in fact fall within this new definition of conditionally essential, however the right conditions to reconstitute growth have not been identified in many cases. Based on this, there is a paramount implication for drug target identification: to be a valid target the deficiencies introduced by a conditionally essential gene must not be overcome during infection. In the case of methionine synthase, it is unlikely that the fungus could acquire sufficient levels of ATP (combined with methionine and not using preferred N-source) in the lung tissue to overcome the growth defect resulting from targeting MetH. The concentration of free extracellular ATP in human plasma has been calculated to be in the sub-micromolar range (28-64 nM) 49. In the lungs, extracellular ATP concentrations must be strictly balanced and increased levels are implicated in the pathophysiology of inflammatory diseases 50; nevertheless, even in such cases ATP levels have been calculated in the low micromolar range 51,52.
We then questioned how the lack of methionine synthase’s enzymatic activity could cause a drop in cell energy. We hypothesised that blockage of methionine synthase activity likely causes a forced conversion of 5,10-methylene-THF to 5-methyl-THF by the action of MetF (Fig. 1A). In support of this, we observed that expression of metF was increased in the H_OFF strain (Fig. 3D). This likely causes a shortage of 5,10-methylene-THF, as the conversion is not reversible and THF cannot be recycled by the action of methionine synthase (Fig. 1A). Indeed, supplementation of folic acid (only when amino acids are the sole N-source, Fig. S3) and of purines could partially restore growth (Fig. 2A), as they compensate for the deficit in purine ring biosynthesis when there is a shortage of 5,10-methylene-THF. However, this still does not explain why there is a drop in ATP. We hypothesised that the block of purine biosynthesis might be sensed as a shortage of nucleotides. This could then cause a shift in glucose metabolism from glycolysis and the TCA cycle (which produce energy) to the Pentose Phosphate Pathway (PPP), which is required to produce ribose-5-phosphate, an integral part of nucleotides. In a similar vein, it has recently been described that activation of anabolism in Saccharomyces cerevisiae implies increased nucleotide biosynthesis and consequently metabolic flow through the PPP 53. To evaluate our hypothesis, we investigated the transcription level of the glucose-6-phosphate dehydrogenase (G6PD) encoding gene (AFUA_3G08470), which catalyses the first committed step of the PPP. In agreement with our hypothesis, the expression of G6PD encoding gene increases in the H_OFF strain upon addition of Dox (Fig. 3D), likely reflecting an increased flow through the PPP. We then wondered how cells may be activating the PPP. The target of rapamycin (TOR) TORC1 effector, which is widely known to activate anabolism and growth 54-56, has been described to activate the PPP in mammalian cells 57,58 and has been functionally connected with energy production and nucleotide metabolism in A. fumigatus 59. In addition, the cAMP/PKA (protein kinase A) pathway is known to be paramount for sensing of nutrients and the correspondent adaptation of gene expression and metabolism 60, and was found to be implicated in the regulation of nucleotide biosynthesis in A. fumigatus 61. Consequently, we explored if a partial block of TOR with low concentrations of rapamycin or of PKA with H-89 could prevent the imbalanced activation of the PPP in the absence of MetH activity. However, neither of the inhibitors could reconstitute growth of the H_OFF strain in restrictive conditions (Fig. S4C). This means that neither the TOR nor the PKA pathways seem to be involved in the deleterious metabolic shift that activates PPP and therefore the mechanism remains to be elucidated.
In summary, we propose that absence of methionine synthase activity causes a strong defect in purine biosynthesis that the cell tries to compensate for by shifting carbon metabolism to the PPP; this metabolic imbalance causes a drop of ATP levels, which collapses cell energetics and results in halted growth (Fig. 3E).
Interestingly, we also detected that metF expression is higher in the H_OFF strain compared to the wild-type, even in the absence of Dox (Fig. 3D). This could be explained as an effort to compensate a higher demand of 5-methyl-THF by the slightly increased amount of methionine synthase in this strain (Fig. S1A). This effect could cause a mild defect in purine biosynthesis in the H_OFF strain, and indeed adenosine content was lower in the H_OFF–Dox condition compared with the wild-type–Dox sample in the metabolome analysis (Fig 3A). Furthermore, this also explains why we detected a small but significant increase of G6PD expression in the H_OFF-Dox condition (Fig. 3D). Therefore, it seems that upregulating methionine synthase has the potential to cause the same metabolic imbalance as downregulating it. However, the effect of overexpression (notice that it is only ∼1.5 fold in our strain Fig. S1A) is minor and does not have obvious consequences for growth, as THF can be recycled and thus the shortage of 5,10-methylene-THF is not severe. In any case, two important points can be highlighted from this small imbalance. Firstly, methionine synthase activity is very important and must be finely tuned to maintain a proper metabolic homeostasis. Secondly, changing the expression level of genes with constitutive and/or regulatable promoters can have unexpected and hidden consequences that often go unnoticed.
Supplementation with S-adenosylmethionine reconstitutes ATP levels and growth
We have shown that the absence of MetH activity causes a reduction in ATP levels. S-adenosylmethionine (SAM) is produced from methionine and ATP by the action of S-adenosylmethionine synthetase SasA (Fig. 1A), an essential enzyme in A. nidulans 62. Hence, we reasoned that the absence of MetH activity might cause a decrease in SAM levels. To test that hypothesis, we first attempted to rescue growth of the H_OFF strain in a medium supplemented with methionine and SAM. We tested various N-sources to diversify the presence of permeases in the cell membrane, aiming to maximise the chances of SAM uptake (Fig. S3). Indeed, the addition of SAM reconstituted growth of the H_OFF strain in restrictive conditions in the presence of methionine when amino acids were the only N-source (Fig. 4A and S3). We then measured the intracellular concentration of SAM in growing mycelia upon addition of Dox using MS/MS. Surprisingly, we observed that addition of Dox to the H_OFF strain did not cause a significant reduction in SAM levels (Fig. 4B). Consequently, we wondered how the addition of SAM may reconstitute H_OFF growth if its levels are not reduced upon metH repression. We speculated that as SAM is a crucial molecule it continues to be produced even if the levels of ATP are reduced, draining it from other cellular processes and thus triggering energy deprivation. In support of this hypothesis, we observed that supplementing SAM to the medium increased the levels of ATP in growing hyphae (Fig. 4C).
In any case, as SAM supplementation can reconstitute H_OFF growth, it constitutes another condition that overcomes the conditional essentiality of metH. However, the concentration of SAM in human serum is extremely low, in the range of 100-150 nM 31, and consequently it is unlikely that the fungus could find sufficient SAM during infection to compensate for the defect in ATP caused by targeting methionine synthase.
S-adenosylmethionine plays a fundamental role as methyl donor for the majority of cellular methylation reactions, including methylation of DNA. Given the observed importance of SAM in the absence of MetH activity and considering that in P. pastoris and C. albicans methionine synthase was reported to localise in the nucleus, as well as in the cytoplasm 34, we speculated that nuclear localization might be important for MetH cellular function. To test this hypothesis, we constructed strains expressing different versions of C-terminus GFP-tagged MetH from the pJA49 plasmid (Fig. S2) in the H_OFF background. These were a wild-type MetH, a MetHD616A (control of no growth –Fig. 1C & S5A–) and a MetHR749A (metHg2439CG>GA) version of the protein, which according to the results published for P. pastoris should not localise in the nucleus 34. The strain expressing wild-type MetH grew normally in restrictive conditions (Fig. S5A), proving that the tagged MetH-GFP protein was active. We confirmed that A. fumigatus MetH localises in both the nucleus and cytoplasm (Fig 4D & S5B). In contrast to what was described in P. pastoris, the MetHR749A protein seems to be active, as it could trigger growth of H_OFF in restrictive conditions (Fig. S5A) and still localised into the nucleus (Fig. S5B). Therefore, the possibility that MetH localisation in the nucleus is important needs further exploration.
Repression of methionine synthase causes growth inhibition in growing mycelia
The major advantage of the tetOFF system is that it can be employed to simulate a drug treatment before a specific chemical is developed. Addition of Dox to a growing mycelium downregulates the gene of interest (Fig S1A), mimicking the effect of blocking its product by the action of a drug. To investigate the effect that blocking MetH has for mycelial growth, we added Dox to 12, 16 or 24 h grown submerged mycelia and left it incubating for an additional 24 h. Addition of Dox to 12 or 16 h grown mycelia severely impaired growth, as observed by biomass (Fig. 5A) and OD (Fig S6A) measurements. This effect was lost when Dox was added to 24 h grown mycelia, due to the incapacity of Dox to reach and downregulate expression in all cells within the dense mass of an overgrown mycelium. Interestingly, Dox addition to methionine free media stopped H_OFF growth immediately, which can be observed by comparing fungal biomass at the time of Dox addition to the measurement 24 h after Dox addition. In contrast, the fungus inoculated in methionine containing media grew a little further after Dox addition (Fig. 5A). To understand this difference, we added Dox to either resting or 8 h germinated conidia and imaged them 16 and 40 hs after drug addition (Fig. 5B & Fig. S6B). In agreement with the previous result, we observed that Dox addition in methionine free medium inhibited growth immediately: resting conidia did not germinate and germinated conidia did not elongate the germtube. In contrast, after addition of Dox in methionine containing medium, most of the resting conidia were still able to germinate and some germlings could elongate the germinated tubes to form short hyphae. This suggests that the drop in ATP levels takes ∼3-4 h before having an effect on growth. Importantly, once growth was inhibited, the effect was sustained for a long period, as we could not detect further growth up to 40 h post-inoculation. To corroborate these observations and further determine whether the effect of growth is fungistatic or fungicidal in the long term, we performed a time-lapse analysis of the effects of adding Dox to 8 h swollen conidia and its subsequent withdrawal after 16 h of incubation (Fig. 5C and Video 1). We observed that growth was inhibited ∼4 h after Dox addition and almost completely halted after 6 h, which was sustained as long as the drug was present. Upon withdrawal of Dox, growth resumes within 6 h (Fig 5D), showing that the effect of blocking MetH is fungistatic, at least with the genetic TeOFF model of metH repression.
Targeting MetH in established infections interferes with the progression of disease
Using the TetON system we previously showed that impeding genetic expression of metH in the infecting conidia resulted in avirulence in a murine model of invasive pulmonary aspergillosis 17. This demonstrated that the murine lung does not readily provide the conditions to overcome the conditional essentiality of metH, and thus this gene is required to establish infection. However, antifungal drugs are normally administered to treat patients who already have an established infection. This could imply that the conditional essentiality of the gene is overcome as the fungal metabolic requirements and the environmental conditions are different when the fungus is actively growing in the tissue. Consequently, in order to achieve a rigorous target validation, it is crucial to assess the efficiency of new target candidates in established infections. Currently, two systems can be used to assess the relevance of fungal essential genes for pulmonary infection: TetON and (p)xylP. These systems can be used to either impede or permit fungal gene expression in murine lungs; but in both models this control must be exerted from the beginning of infection, as sufficient levels of the inducing molecule (doxycycline or xylose) must be present to activate gene expression in the control condition. Consequently, these models have been used to investigate the role of genes that are required to grow in vitro to establish infection 17,63,64. However, those models cannot be used to mimic drug treatments of already established aspergillosis infections in vivo. Therefore, we aimed to optimise the use of the TetOFF system for this purpose, as it can be used to downregulate gene expression in growing mycelia. As a control for the model, we constructed a cyp51A_tetOFFΔcyp51B (51A_OFF) strain. We reasoned that the target of the azoles, first-line treatment drugs for Aspergillus diseases, should be the gold standard to compare to for any target. This strain showed a similar behaviour as H_OFF in vitro: as little as 0.05 µg/ml Dox prevented colony development on an agar plate (Fig. S7A) and addition of Dox to conidia or germlings blocked growth (Fig. S7B).
We first assayed the use of the TetOFF system in the Galleria mellonella alternative mini-host model of infection. Preliminary experiments revealed that the balance between reaching sufficient levels of Dox to exert an effect and preventing toxic effects of overdose was very delicate. We finally optimised a regimen consisting of 5 injections of 50 mg/kg Dox (Fig. S8A). We then infected Galleria larvae with 5×102 conidia of 51A_OFF or H_OFF strains and applied the Dox regimen or PBS vehicle starting at the same time of infection (0 h) or 6 h after infection (Fig. S8A). For both strains, administration of Dox from the beginning of infection triggered a significant improvement in survival compared with the non-treated conditions (50% VS 17.2% for 51A_OFF, P=0.0036, and 41.45% VS 6.67% for H_OFF, P=0.022) (Fig. 6A). Notably, the administration of Dox at the time of infection, with either strain, did not improve survival to close to 100%. Considering that both strains are unable to grow in the presence of the drug (Fig. S6B, S7B), this was a surprising result that suggests the levels of Dox reached in vivo are not sufficient to completely downregulate gene expression. This could be due to a rapid metabolization of the drug in the larvae hemocoel or to microenvironment variations in its concentration. Despite this caveat of the model, we observed that administration of Dox 6 h after infection also triggered a significant improvement in survival for both strains (42.8% VS 17.2% for 51A_OFF, P=0.0007, and 32.26% VS 6.67% for H_OFF, P=0.0324) (Fig. 6A). Therefore, downregulation of methionine synthase genetic expression in established infections conferred a significant benefit in survival which was comparable to that observed with the target of the azoles.
The positive results obtained using the Galleria infection model prompted us to assay the TetOFF system in a leukopenic murine model of pulmonary aspergillosis. To ensure that Dox levels in mouse lungs reach and maintain sufficient concentrations to downregulate gene expression (according to our results in vitro) we performed a pilot Dox dosage experiment in immunosuppressed non-infected mice (Fig S8B). We extracted lungs of Dox treated mice at different time-points, homogenated them and measured Dox concentration using a bioassay based on inhibition of Escherichia coli DH5α growth. We could detect promising levels of Dox in all mice (concentrations ranging from 2.2 to 0.94 µg/mL –Fig. S8B–) which according to our results in vitro should be sufficient to downregulate gene expression from the TetOFF system. We therefore infected leukopenic mice with 105 spores of the 51A_OFF or the H_OFF strains and administered PBS vehicle or our Dox regimen, starting 16 h after infection (Fig. S8B). The use of an uninfected, Dox treated control group uncovered that the intense Dox regimen used was harmful for the mice. These uninfected mice lost weight at a similar rate as the infected groups and looked ill from the third or fourth day of treatment. This might be due to a toxic effect caused by the previously described iron chelating properties of Dox 65, although we have made no attempt to confirm this. As a consequence, there was no beneficial effect of Dox treatment on survival (not shown). The fact that Dox treatment did also not show any benefit in survival for our control strain 51A_OFF, which should mimic treatment with azoles (primary therapy for invasive aspergillosis), indicates that the TetOFF system is not adequate to mimic a drug treatment in established infections. Nevertheless, we further attempted to determine the efficiency of targeting MetH in established infections by measuring fungal burdens in lungs of treated and untreated mice. We observed that two and a half days of Dox treatment (which had not caused visible toxic effects) did result in a significant reduction of fungal burdens 3 days after infection for both 51A_OFF (P=0.0279) and H_OFF (P=0.0019) (Fig. 6B). Therefore, we could observe a beneficial effect of interfering with methionine synthase genetic expression in an established pulmonary infection, which was comparable to that of interfering with the expression of cyp51A, the target of azoles. To our knowledge, this is the most rigorous validation made for a fungal target in vivo to date.
A recent study also aimed to use another TetOFF system to validate a drug target in established aspergillosis infections 66. These authors administered Dox exclusively through oral gavage, accounting for lower dosage of drug. Consequently, even if no toxic effect was observed, they did also not detect any beneficial effect on survival when the Dox treatment was initiated after infection. Therefore, even if the TetOFF system is currently the only model that allows investigating the efficiency of new targets in aspergillosis established infections, it is clearly not optimal and better models are needed.
Structural-based virtual screening of MetH
Having shown in vivo that MetH is a promising target, we decided to investigate its druggability by running a structural-based virtual screening. The sequence of A. fumigatus MetH (AfMetH) contains two predicted methionine synthase domains with a β-barrel fold conserved in other fungal and bacterial enzymes. The structure of the C. albicans orthologue 67 (CaMetH) showed that the active site is located between the two domains where the methyl tetrahydrofolate, the homocysteine substrate and the catalytic zinc ion bind in close proximity. The homology model for AfMetH (Fig. 7A) overlaps very well with that of the CaMetH thus providing a suitable molecular model for further analysis. In contrast, the structure of the human methionine synthase (hMS) shows a very different overall arrangement with the folate and homocysteine binding domains located in completely different regions (Fig. 7B). Comparison of the tetrahydrofolate binding sites between the fungal and the human structures also highlights significant structural differences that affect the conformation adopted by the ligand. In the CaMetH structure the 5-methyl-tetrahydrofolate (C2F) adopts a bent conformation (<20Å long) and it is in close proximity to the methionine product, whereas in the human structure the tetrahydrofolate (THF) ligand binds in an elongated conformation extending up to 30Å from end to end, (Fig. 7 C&D).
Virtual screening (VS) was carried on the AfMetH and the hMS structures with the Maybridge Ro3 fragment library to explore potential venues for drug development. The results showed four ligand binding clusters in the AfMetH structure, two of which (C1, C2) match the binding position of the 5-methyl-tetrahydrafolate and the methionine from the CaMetH crystal structure (Fig. 7E). For the hMS, we found two main clusters, C1 that overlaps with the tetrahydrofolate binding site and C2 in a nearby pocket. Clearly the distribution of the clusters defines a very different landscape around the folate site between the human and the fungal enzymes. Furthermore, the proximity of the C1 and C2 clusters, matching the folate and Met/homocysteine binding sites in the Ca/Af proteins means that it may be possible to combine ligands at both sites to generate double-site inhibitors with high specificity towards the fungal enzymes. Antifolates are a class of drugs that antagonise folate, blocking the action of folate dependent enzymes such as dihydrofolate reductase (DHFR), thymidylate synthase or methionine synthase. Methotrexate is an antifolate commonly used to treat cancer and autoimmune diseases. Interestingly, methotrexate has been shown to be a weak inhibitor of the C. albicans methionine synthase 33 and to have some antifungal activity against C. albicans 68 and Aspergillus ssp 69. Nevertheless, methotrexate is not a good antifungal drug, as its activity is high against human enzymes (IC50 of 0.3 µM for DHFR 70) and low against fungal methionine synthase (IC50 of 4 mM for C. albicans MetH 33). Therefore, more potent and specific inhibitors of fungal methionine synthases are needed to fully exploit the value of this target for antifungal therapy, a task that seems possible and can be directed from our analyses.
In summary, we have shown that methionine synthase blockage triggers not only methionine auxotrophy, but also a metabolic imbalance that results in a drop in cellular energetics and growth arrest. In light of our results, we propose that conditional essentiality is important to understand the underlying mechanisms of metabolic processes and needs to be considered to achieve proper validation of novel antimicrobial targets. Accordingly, we proved that targeting methionine synthase in established infections has a beneficial effect similar to that observed for the target of azoles, the most effective drugs for the treatment of aspergillosis. Finally, we showed that fungal methionine synthases have distinct druggable pockets that can be exploited to design specific inhibitors. In conclusion, we have demonstrated that fungal methionine synthases are promising targets for the development of novel antifungals
MATERIAL AND METHODS
Strains, media and culture conditions
The Escherichia coli strain DH5α 71 was used for cloning procedures. Plasmid-carrying E. coli strains were routinely grown at 37°C in LB liquid medium (Oxoid) under selective conditions (100 µg·mL-1 ampicillin or 50 µg·mL-1 kanamycin); for growth on plates, 1.5% agar was added to solidify the medium. All plasmids used in the course of this study were generated using the Seamless Cloning (Invitrogen) technology as previously described 17,72. E. coli strain BL21 (DE3) 73 was grown on Mueller Hinton agar (Sigma) in bioassays, to determine Dox concentrations within homogenized murine lungs, as previously described by Law and colleagues 74.
The wild-type clinical isolate Aspergillus fumigatus strain ATCC 46645 served as reference recipient. A. fumigatus strain A1160 (ku80Δ) 75 was also used to confirm metH essentiality. A. fumigatus mutants were generated using a standard protoplasting protocol 76. A. fumigatus strains were generally cultured in minimal medium (MM) 77 (1% glucose, 5 mM ammonium tartrate, 7 mM KCl, 11 mM KH2PO4, 0.25 mM MgSO4, 1× Hutner’s trace elements solution; pH 5.5; 1.5% agar) at 37°C. For selection in the presence of resistance markers 50 µg·mL-1 of hygromycin B or 100 µg·mL-1 of pyrithiamine (InvivoGen) were applied. In sulfur-free medium (MM-S), MgCl2 substituted for MgSO4 and a modified mixture of trace elements lacking any sulfate salt was used. For all growth assays on solid media, the culture medium was inoculated with 10 µl of a freshly prepared A. fumigatus spore suspension (105 conidia·mL-1 in water supplemented with 0.9% NaCl and 0.02% Tween 80) and incubated at 37°C for 3 days.
Extraction and manipulation of nucleic acids
Standard protocols of recombinant DNA technology were carried out 78. Phusion® high-fidelity DNA polymerase (ThermoFisher Scientific) was generally used in polymerase chain reactions and essential cloning steps were verified by sequencing. Fungal genomic DNA was prepared following the protocol of Kolar et al. 79 and Southern analyses were carried out as described 80,81, using the Amersham ECL Direct Labeling and Detection System® (GE Healthcare). Fungal RNA was isolated using TRIzol reagent (ThermoFisher Scientific) and Qiagen plant RNA extraction kit. Retrotranscription was performed using SuperScript III First-Strand Synthesis (ThermoFisher Scientific). RT-PCR on both gDNA and cDNA was performed using the SYBR® Green JumpStart (Sigma) in a 7500 Fast Real Time PCR cycler from Applied Biosystems.
Microscopy
103 A. fumigatus resting or 8 h germinated conidia were inoculated in 200 µL of medium (+/- Dox) in 8 well imaging chambers (ibidi) and incubated at 37°C. Microscopy images were taken on a Nikon Eclipse TE2000-E, using a CFI Plan Apochromat Lambda 20X/0.75 objective and captured with a Hamamatsu Orca-ER CCD camera (Hamamatsu Photonics) and manipulated using NIS-Elements 4.0 (Nikon). For extensively grown mycelia a stereomicroscope Leica MZFL-III was used, with a Q-imaging Retinga 6000 camera, and manipulated using Metamorph v7760. Confocal imaging was performed using a Leica TCS SP8x inverted confocal microscope equipped with a 40X/0.85 objective. Nuclei were stained with DAPI (Life Technologies Ltd) as described previously74. GFP was excited at 458 nm with an Argon laser at 20% power. DAPI was excited at 405 nm with an LED diode at 20%.
Metabolome analyses
A. fumigatus wild-type and metH_tetOFF strains were incubated in MM for 16 h before the -Dox samples were taken (8 replicates of 11 mL each). Then, 5 µg/mL Dox and 5 mM methionine (to prevent metabolic adaptation due to met auxotrophy) were added as appropriate and the cultures incubated for 6 h, after which the +Dox samples were taken (8 × 11 mL). The samples were immediately quenched with 2× volumes of 60% methanol at -48°C. After centrifugation at 4800 g for 10 min at -8°C, metabolites were extracted in 1 mL 80% methanol at -48°C by three cycles of N2 liquid snap freezing, thawing and vortexing. Supernatant was cleared by centrifugation at -9 °C, 14,500 g for 5 min. Quality control (QC) samples were prepared by combining 100 µL from each sample. Samples were aliquoted (300 µL), followed by the addition of 100 µL of the internal standard solution (0.2 mg/mL succinic-d4 acid, and 0.2 mg/mL glycine-d5) and vortex mix for 15 s. All samples were lyophilised by speed vacuum concentration at room temperature overnight (HETO VR MAXI vacuum centrifuge attached to a Thermo Svart RVT 4104 refrigerated vapour trap; Thermo Life Sciences, Basingstoke, U.K.). A two-step derivatization protocol of methoxyamination followed by trimethylsilylation was employed 82.
GC-MS analysis was conducted on a 7890B GC coupled to a 5975 series MSD quadrupole mass spectrometer and equipped with a 7693 autosampler (Agilent, Technologies, UK). The sample (1 μL) was injected onto a VF5-MS column (30 m × 0.25 mm × 0.25 μm; Agilent Technologies) with an inlet temperature of 280 °C and a split ratio of 20:1. Helium was used as the carrier gas with a flow rate of 1 mL/min. The chromatography was programmed to begin at 70 °C with a hold time of 4 min, followed by an increase to 300 °C at a rate of 14 °C/min and a final hold time of 4 min before returning to 70 °C. The total run time for the analysis was 24.43 min. The MS was equipped with an electron impact ion source using 70 eV ionisation and a fixed emission of 35 μA. The mass spectrum was collected for the range 50-550 m/z with a scan speed of 3,125 (N=1). Samples were analysed in a randomised order with the injection of a pooled biological quality control sample after every 6th sample injection.
For data analysis, the GC-MS raw files were converted to mzXML and subsequently imported to R. The R package “erah” was employed to de-convolve the GC-MS files. Chromatographic peaks and mass spectra were cross-referenced with the Golm library for putative identification purposes, and followed the metabolomics standards initiative (MSI) guidelines for metabolite identification 83. The peak intensities were normalised according to the IS (succinic-d4 acid) before being log10-scaled for further statistical analysis. All pre-processed data were investigated by employing principal component analysis (PCA) 84.
The raw data of this metabolome analysis has been deposited in the MetaboLights database 85, under the reference MTBLS1636 (www.ebi.ac.uk/metabolights/MTBLS1636)
ATP Quantitation
A. fumigatus was grown as in the metabolome analysis. However, where the effect of SAM was investigated spores were inoculated into MM-N + 1mg/mL aac and 0.5mM SAM was also added at the time of Dox addition. ATP levels were determined using the BacTiter-Glo™ Assay (Promega) following the manufacturer’s instructions and a TriStar LB 941 Microplate Reader (Berthold).
Isolation and detection of SAM
A. fumigatus was grown exactly in the same conditions as described for the metabolome analysis. Harvested mycelia were snap-frozen in liquid N2 and stored at -70 °C before SAM isolation. SAM extraction was carried out according to 86. Briefly, frozen mycelia were ground in liquid N2 and 0.1 M HCl (250 µL) was added to ground mycelia (100 mg). Samples were stored on ice for 1 h, with sample vortexing at regular intervals. Samples were centrifuged at 13,000 g for 10 min (4 °C) to remove cell debris and supernatants were collected. Concentration of protein in supernatants was determined using a Biorad Bradford protein assay relative to a bovine serum albumin (BSA) standard curve. Clarified supernatants were adjusted to 15 % (w/v) trichloroacetic acid to remove protein. After 20 min incubation on ice, centrifugation was repeated and clarified supernatants were diluted with 0.1 % (v/v) formic acid. Samples were injected onto a Hypersil Gold aQ C18 column with polar endcapping on a Dionex UltiMate 3000 nanoRSLC with a Thermo Q-Exactive mass spectrometer. Samples were loaded in 100 % Solvent A (0.1 % (v/v) formic acid in water) followed by a gradient to 20 % B (Solvent B: 0.1 % (v/v) formic acid in acetonitrile) over 4 min. Resolution set to 70000 for MS, with MS/MS scans collected using a Top3 method. SAM standard (Sigma) was used to determine retention time and to confirm MS/MS fragmentation pattern for identification. Extracted ion chromatograms were generated at m/z 399-400 and the peak area of SAM was measured. Measurements were taken from three biological and two technical replicates per sample, normalized to the protein concentration in the extracts from each replicate. SAM levels are expressed as a percentage relative to the parental strain in the absence of Dox.
Biomass measurement
Conidia were inoculated into MM-S, supplemented with either methionine or sulfate, and incubated at 37°C 180 rpm for 12, 16 or 24 h. After this initial incubation, 3 mL samples were taken in triplicate from the cultures, filtered through tared Miracloth, dried at 60°C for 16 h and their biomass measured. In treated conditions Dox was added to a final concentration of 1 μg/mL and the culture allowed to grow for a further 24 h at 37°C 180 rpm. 5 mL samples were taken in triplicate and their biomass measured as above.
Galleria mellonella infections
Sixth-stage instar larval G. mellonella moths (15 to 25 mm in length) were ordered from the Live Foods Company (Sheffield, United Kingdom). Infections were performed according to Kavanagh and Fallon 87. Randomly selected groups of 15 larvae were injected in the last left proleg with 10 µL of a suspension of 5×104 conidia/mL in PBS, using Braun Omnican 50-U 100 0.5-mL insulin syringes with integrated needles. Dox was administered according to the treatment shown in Fig. S7A, alternating injections in the last right and left prolegs. In each experiment an untouched and a saline injected control were included, to verify that mortality was not due to the health status of the larvae or the injection method. Three independent experiments were carried out. The presented survival curves display the pooled data, which was analysed with the Log-Rank test.
Leukopenic murine model of invasive pulmonary aspergillosis and calculation of fungal burden
All experiments were performed under United Kingdom Home Office project license PDF8402B7 and approved by the University of Manchester Ethics Committee. Outbred CD1 male mice (22– 26 g) were purchased from Charles Rivers and left to rest for at least 1 week before the experiment. Mice were allowed access ad libitum to water and food throughout the experiment. Mice were immunosuppressed with 150 mg/kg of cyclophosphamide on days -3 and -1 and with 250 mg/kg cortisone acetate on day -1. On day 0 mice were anesthetized with isofluorane and intranasally infected with a dose of 105 conidia (40 µL of a freshly harvested spore solution of 2.5 × 106 conidia/mL). Dox was administered according to the treatment shown in Fig. S7B. Dox containing food was purchased from Envingo (Safe-diet U8200 Version 0115 A03 0.625 g/kg Doxycycline Hyclate pellets). At the selected time-point (72 h after infection for fungal burden) mice were sacrificed by a lethal injection of pentobarbital, the lungs harvested and immediately frozen.
Frozen lungs were lyophilised for 48 h in a CoolSafe ScanVac freeze drier connected to a VacuuBrand pump and subsequently ground in the presence of liquid nitrogen. DNA was isolated from the powder using the DNeasy Blood & Tissue Kit (Qiagen). DNA concentration and quality was measured using a NanoDrop 2000 (ThermoFisher Scientific). To detect the fungal burden, 500 ng of DNA extracted from each infected lung were subjected to qPCR. Primers used to amplify the A. fumigatus β-tubulin gene (AFUA_7G00250) were forward, 5’-ACTTCCGCAATGGACGTTAC-3’, and reverse, 5’-GGATGTTGTTGGGAATCCAC-3’. Those designed to amplify the murine actin locus (NM_007393) were forward, 5’-CGAGCACAGCTTCTTTGCAG-3’ and reverse, 5’-CCCATGGTGTCCGTTCTGA-3’. Standard curves were calculated using different concentrations of fungal and murine gDNA pure template. Negative controls containing no template DNA were subjected to the same procedure to exclude or detect any possible contamination. Three technical replicates were prepared for each lung sample. qPCRs were performed using the 7500 Fast Real-Time PCR system (Thermo Fisher Scientific) with the following thermal cycling parameters: 94 °C for 2 min and 40 cycles of 94°C for 15 s and 59°C for 1 min. The fungal burden was calculated by normalising the number of fungal genome equivalents (i.e. number of copies of the tubulin gene) to the murine genome equivalents in the sample (i.e number of copies of the actin gene) 88. Two independent experiments were carried out (n=9, 5 mice in the first and 4 mice in the second experiment). Burdens for each strain were compared using a Mann Whitney test.
Molecular homology models and virtual screening
The full-length sequence for AFUA_4G07360, the cobalamin-independent methionine synthase MetH from A. fumigatus (AfMetH) was obtained from FungiDB (https://fungidb.org/fungidb) 89. This sequence together with the structure of the C. albicans orthologue (CaMetH) (PDB ID: 4L65, DOI: 10.1016/j.jmb.2014.02.006) were used to create the molecular homology model in Modeller (version 9.23) 90 with the basic option mode. The AfMetH model was then used for virtual screening with the semi-automated pipeline VSpipe 91. For comparison we also performed virtual screening with the structure of the human methionine synthase (hMS) containing the folate and homocysteine binding domains (PDB ID: 4CCZ). Docking was done using the Maybridge Ro3 1000 fragment library with AutoDock Vina 92. Results were inspected graphically using PyMol (v1.8.0.3 Enhanced for Mac OS X (Schrödinger). All images were produced with PyMol.
Nuclei isolation
Protoplasts were generated as in A. fumigatus transformations 76 and nuclei isolated were isolated by sucrose gradient fractionation as previously described by Sperling and Grunstein 93. Nuclear localisation of GFP-tagged target proteins was confirmed by Western-blot. Aliquots of nuclei were boiled for 5 minutes in loading buffer (0.2 M Tris-HCl, 0.4 M DTT, 8% SDS, trace bromophenol blue) and separated on a 12% (w/v) SDS-PAGE gel. The proteins were transferred to a Polyvinylidene difluoride (PVDF) membrane using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). Detection of GFP was carried out with a rabbit polyclonal anti-GFP antiserum (Bio-Rad) and anti-rabbit IgG HRP-linked antibody (Cell Signalling Technology). SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) and the ChemiDoc XRS+ Imaging System (Biorad) were used to visualise immunoreactive bands. Ponceau S staining was performed to normalize the Western-blot signal to the protein loading.
AUTHOR CONTRIBUTION
JS performed the majority of experiments, analysed and interpreted most of the data and participated in the design of the project. MS helped with the acquisition and analysis of most of the experiments. BT run the structural-based virtual screening. RAO measured SAM levels in mycelia. HMA performed the metabolomic experiment, analysed and with RG interpreted the data. RFG assisted with the mouse models of infection. RT helped with the execution of qPCRs. KH helped to set up the GC-MS instrument. SD designed the MS/MS analysis of SAM. RG designed the metabolome analysis and interpreted the data. LT designed the virtual screening analysis and interpreted the data. EB participated in the design and conception of the project. JA conceived and designed the project and analysed most of the data.
COMPETING INTERESTS
The authors declare no competing interests.
DATA AVAILABILITY
The raw data that support the findings of this study are available upon reasonable request to the authors. The raw data of metabolome analysis has been deposited in the MetaboLights database 85, under the reference MTBLS1636 (www.ebi.ac.uk/metabolights/MTBLS1636).
ACKNOWLDGEMENTS
We acknowledge the use of the Phenotyping Center at Manchester (PCAM) for the use of their microscopes and advanced image analysis workstations. We are grateful to Prof Sven Krappmann for critical reading of the manuscript and his constant support. We would like to thank all members of MFIG for constant help and encouragement.
JA was supported by a MRC Career Development Award (MR/N008707/1). JS was supported by a BSAC scholarship (bsac-2016-0049). BPT was supported by a MRC Doctoral Training Partnership PhD studentship. Q-Exactive mass spectrometer and nanoLC instrumentation were funded by a competitive infrastructure award from Science Foundation Ireland (SFI) (12/RI/2346(3)).