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. 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 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.

worldwide. The current repertoire of antifungal drugs is very limited and, worryingly, resistance 48 has emerged and already become a serious threat to our capacity to treat fungal diseases. The 49 first step to develop new drugs often is to identify molecular targets which inhibition during 50 infection can prevent pathogen growth. However, the current models are not suitable to 51 validate targets in established infections. Here we have characterised the promising antifungal 52 target methionine synthase in great detail, using the prominent fungal pathogen Aspergillus 53 fumigatus as a model. We have uncovered the underlying reason for its essentiality and 54 confirmed its druggability. Furthermore, we have optimised the use of a genetic system to show 55 a beneficial effect of targeting methionine synthase in established infections. Therefore, we 56 believe that antifungal drugs to target methionine synthase should be pursued and additionally, 57 we propose that antifungal targets should be validated in a model of established infection. 58 59 60 61 62 INTRODUCTION metH_tetOFF strains (H_OFF) grew as the wild type in the absence of Dox, but as little as 0.5 130 µg/mL was sufficient to completely prevent colony development on an agar plate even in the 131 presence of methionine (Fig. S1B). This corroborates our previous result that methionine 132 synthase is essential for A. fumigatus viability and that its absence does not result in a sheer 133 auxotrophy for methionine (17). 134 Methionine synthase forms an interjection between the trans-sulfuration pathway and the 135 one carbon metabolic route (Fig. 1A), as the enzyme utilizes 5-methyl-tetrahydrofolate as co-136 substrate. Therefore, the essentiality of metH might be due to required integrities of the trans-137 sulfuration pathway or of the one carbon metabolic route. Alternatively, it could be that the 138 presence of the enzyme itself is essential, either because its enzymatic activity is required or 139 because it is fulfilling an unrelated additional role, as being part of a multiprotein complex. To 140 start discerning among these possibilities, we constructed a double ΔmetGΔcysD mutant, 141 blocked in the previous step of the trans-sulfuration pathway, and a ΔmetF deletant, which 142 blocks the previous step of the one-carbon metabolic route (Fig. 1A). As we had previously 143 observed (26), to rescue fully metF's growth the media had to be supplemented with 144 methionine and other amino acids, as the folate cycle is necessary for the interconversion of 145 serine and glycine and plays a role in histidine and aromatic amino acid metabolism (27,28). 146 Consequently, we added a mix of all amino acids except cysteine and methionine to the S-free 147 medium for this experiment. Phenotypic tests (Fig. 1B) confirmed that the ΔmetGΔcysD and 148 metF mutants were viable and could grow in the presence of methionine. In contrast, the 149 H_OFF conditional strain could not grow under restrictive conditions even in the presence of the 150 amino acid mix and methionine (Fig. 1B). Therefore, the MetH protein itself, and not the integrity 151 of the trans-sulfuration and one-carbon pathways, is essential for A. fumigatus viability. 152 Interestingly, the methionine auxotroph ΔmetGΔcysD was avirulent in a leukopenic model of 153 pulmonary aspergillosis (Fig. S1C), suggesting that the amount of readily available methionine 154 in the lung is very limited, not sufficient to rescue its auxotrophy. Indeed, the level of methionine 155 in human serum was calculated to be as low as ~20 µM (29, 30), which was described as 156 insufficient to support the growth of various auxotrophic bacterial pathogens (31) and we have 157 also observed that is not enough to rescue growth of the A. fumigatus ΔmetGΔcysD auxotroph. 158 Essentiality of the MetH protein could be directly linked to its enzymatic activity or, 159 alternatively, the protein could be performing an additional independent function. To discern 160 between these two possibilities, we constructed two strains that express single-point mutated 161 versions of MetH from the innocuous Ku70 locus of the H_OFF background strain, under the 162 control of its native promoter (Fig. 1C). These point mutations, metH g2042A>2C (D616A) and 163 metH g2179TA>9GC (Y662A), were previously described to prevent conformational rearrangements 164 required for activity of the C. albicans methionine synthase (32). In the absence of Dox, these 165 strains grew normally, as they expressed both the wild-type MetH, from the tetOFF promoter, 166 and the mutated version of the protein (Fig. 1C). In the presence of Dox, when the wild-type 167 metH gene was downregulated, the Y662A strain grew on sulfate worse than in non-restrictive 168 conditions, but still to a significant extent, suggesting that this point mutation did not completely 169 abrogate enzymatic activity (Fig. 1C). Interestingly, Y662 grew normally on methionine, showing 170 that methionine can compensate for a partial reduction of MetH activity (Fig 1C). The D616A 171 mutated protein was confirmed to be stable as a GFP-tagged version of this protein could be 172 visualised in -Dox conditions (Fig. S1D, strain detailed later in the manuscript). Interestingly, the 173 D616A strain (two isolates were tested) was not able to grow on sulfate (Fig. 1C), demonstrating 174 that enzymatic activity was fully blocked. Nor could it grow on methionine (Fig. 1C), indicating 175 that enzymatic activity is required for viability even in the presence of the full protein. All these 176 phenotypes support the conclusion that methionine synthase enzymatic activity is required for 177 viability. 178 179 Absence of methionine synthase enzymatic activity results in a shortage of crucial metabolites, 180 but does not cause toxic accumulation of homocysteine 181 The absence of methionine synthase enzymatic activity has two direct consequences, which 182 could cause deleterious effects and therefore explain its essentiality (Fig. 1A). It could cause an 183 accumulation of the potentially toxic substrate homocysteine and/or a shortage of the co-184 product tetrahydrofolate (THF). THF is directly converted to 5,10-methylene-THF, which is 185 required for the synthesis of purines and thymidylate (TMP), and thus for DNA synthesis; 186 additionally, as purine biosynthesis requires Gln, Gly and Asp, and THF de novo synthesis 187 requires chorismate (precursor of aromatic amino acids), a shortage of THF might cause a 188 depletion of amino acids (Fig. 1A). To investigate if the depletion of any of these metabolites 189 underlies MetH essentiality, we supplemented the media with a number of precursors and 190 potentially depleted metabolites ( Fig. 2A). Added as sole supplement, only adenine was able to 191 trigger growth, but to a minimal degree. Further addition of a mixture of all amino acids 192 noticeably improved growth. Supplementation with adenine and guanine (purine bases) did also 193 reconstitute noticeable growth, which was not enhanced with further addition of amino acids. 194 Folic acid was also capable of reconstituting growth, but only when amino acids were added as 195 the sole N-source (Fig. S3). However, no combination of compounds was able to reconstitute 196 growth to the wild-type level. This suggests that a shortage of relevant metabolites derived from 197 THF, prominently adenine, partially accounts for methionine synthase essentiality, but cannot 198 explain it completely. In other fungi, as Pichia pastoris (33) or Schizosaccharomyces pombe (34), 199 summplementation with methionine and adenine was found to restore growth of a metH 200 mutant to wild-type levels, denoting them as combined auxotrophs. In A. fumigatus it seems to 201 be more complex because supplementation with methionine, adenine and other amino acids 202 could still not fully restore growth, suggesting that more factors are implicated. 203 To investigate if homocysteine could be accumulating to toxic levels in the absence of MetH 204 activity, we over-expressed several genes that should alleviate its accumulation. To this aim we 205 designed and constructed the plasmid pJA49, which allows direct integration of any ORF to 206 episomally overexpress genes in A. fumigatus. Plasmid pJA49 carries the A. nidulans AMA1 207 autologous replicating sequence (35, 36) and the hygromycin B resistance gene (hygrB) as a 208 selection marker. A unique StuI restriction site allows introduction of any PCR amplified ORF in 209 frame under the control of the A. fumigatus strong promoter hspA (37) and the A. nidulans trpC 210 terminator ( Fig S2A). Using this plasmid, we produced a strain in the H_OFF background that 211 episomally overexpresses mecA, encoding cystathionine-β-synthase, which converts 212 homocysteine to cystathionine (Fig. 1A). Homocysteine exerts toxic effects through its 213 conversion to S-adenosylhomocysteine, which causes DNA hypomethylation (38, 39), or to 214 homocysteine thiolactone, which causes N-homocysteinylation at the ε-amino group of protein 215 lysine residues (40, 41). Consequently, we also constructed strains that episomally over-express 216 genes that could detoxify those products: the S-adenosyl-homocysteinase lyase encoding gene 217 sahL (AFUA_1G10130) or the A. nidulans homocysteine thiolactone hydrolase encoding gene 218 blhA (AN6399) (A. fumigatus genome does not encode any orthologue) (Fig. S2B). However, 219 despite a strong over-expression of the genes (Fig. S2C&D), none of them could rescue growth 220 of the H_OFF strain in restrictive conditions (Fig. 2B). Therefore, our over-expression 221 experiments suggest that homocysteine accumulation is not responsible for metH essentiality, 222 but additional experiments such as quantification homocysteine levels, which are currently 223 challenging, would be required to further support this hypothesis. Addition of adenine to the 224 medium did not improve growth of the overexpression strains further than that of the H_OFF 225 background (Fig. 2B), indicating that methionine synthase essentiality seemingly is not a 226 combined effect of homocysteine accumulation and depletion of THF-derived metabolites. Toxic 227 accumulation of homocysteine was speculated to be the underlying reason of methionine 228 synthase essentiality in both Candida albicans and Cryptococcus neoformans (19, 20) but our 229 results suggest that this is not the case in A. fumigatus. Therefore, we propose that the previous 230 assumption should be revisited in other fungal pathogens. 231 232

Methionine synthase repression triggers a metabolic imbalance that causes a decrease in cell 233
energetics 234 Aiming to identify any adverse metabolic shift in the absence of MetH and/or accumulation of 235 toxic compounds that could explain its necessity for proper growth, we performed a 236 metabolomics analysis, via gas chromatography-mass spectrometry (GC-MS), comparing the 237 metabolites present in wild-type and H_OFF strains before and 6 h after Dox addition. Before 238 Dox addition both strains clustered closely together in a Principal Component Analysis (PCA) 239 scores plot (Fig. S4A), showing that their metabolic profiles are highly similar. However, 6 h after 240 Dox addition the strains clusters became clearly separated, denoting differential metabolite 241 content. Analysis of the differentially accumulated metabolites (full list can be consulted in Table  242 S1) using the online platforms MBRole (42) and Metaboanalyst (43, 44) did not reveal any 243 obvious metabolic switch, probably due to the rather small number of metabolites that could 244 be identified by cross-referencing with the Golm library (http://gmd.mpimp-golm.mpg.de/). 245 Manual inspection of the metabolites pointed out interesting aspects. Firstly, the methionine 246 levels were not significantly different, which demonstrates that methionine supplementation in 247 the growth medium triggers correct intracellular levels in the H_OFF strain; this undoubtedly 248 rules out that a shortage of methionine could be the cause of the essentiality of methionine 249 synthase. Secondly, we detected a significantly lower amount of adenosine in the H_OFF strain 250 compared with the wild-type after Dox addition (Fig. 3A), which is in agreement with our 251 previous result that supplementation of adenine can partially reconstitute growth in the 252 absence of MetH. We did not find accumulation of compounds with a clear toxic potential upon 253 metH repression. Nevertheless, we detected a lower amount of several amino acids (Phe,Ser,254 Glu, Pro, Ile, Thr, Ala and Asp, Fig. S4B), which suggests that the cells may enter into growth 255 arrest upon metH repression. Interestingly, we noticed a significantly lower accumulation of 256 some metabolites of the glycolysis pathway and TCA cycle (Fig. 3A) and some other mono and 257 poly-saccharides (Fig. S4B). These variations could reflect a low energetic status of the cells upon 258 metH repression. Indeed, we found that the level of ATP significantly decreased in the H_OFF 259 strain, but not in the wild-type, upon Dox addition (Fig. 3B). Therefore, we evaluated if 260 supplementation of the medium with substrates that have the potential to increase cell 261 energetics can rescue H_OFF growth in restrictive conditions. We found that when pyruvate, 262 which can directly be converted to acetyl-CoA to enter the TCA cycle, was added as the sole 263 carbon source H_OFF growth was reconstituted in restrictive conditions to the same level as the 264 wild-type (Fig. 3C). Growth was limited for both strains, as pyruvate does not appear to be a 265 good carbon source (45). However, the presence of glucose in the medium precluded the 266 reconstitution of growth of H_OFF (Fig. S3), as it has been described to prevent pyruvate uptake 267 in S. cerevisiae (46). We next tested the capacity of ATP to be used an alternative energy source 268 and to reconstitute growth. To diversify the presence of permeases in the cell membrane, and 269 thus maximise the chance of ATP uptake, we assayed two different N-sources: ammonium (NH4 + , 270 preferred source) and amino acids (Fig. S3). Indeed, when amino acids were the only N-source, 271 supplementation of the medium with ATP reconstituted H_OFF growth in restrictive conditions 272 to wild type levels (Fig. 3C). This agrees with the recent observation that eukaryotic cells can 273 uptake ATP and exploit it as an energy source (47). In conclusion, a decrease in cell energetics 274 developed in the absence of methionine synthase seems to explain MetH essentiality for 275 growth. 276 The fact that growth in the absence of methionine synthase can be reconstituted when there 277 are sufficient levels of methionine and ATP implies that metH is a conditionally essential gene, 278 meaning that it is only essential in the absence of the specific conditions that overcome the 279 disturbances derived from its deficiency. We believe that a significant number of genes 280 previously described as essential in fungi would in fact be conditionally essential, however the 281 right conditions to reconstitute growth have not been identified in many cases. This highlights a 282 paramount consideration for the proper identification and validation of drug targets: the 283 deficiencies introduced by targeting a conditionally essential gene must not be overcome during 284 infection. This important concept has already been discussed by others (48-51) and we believe 285 addressing it should become the standard for proper validation of antimicrobial targets. In the 286 case of methionine synthase, it is unlikely that the fungus could acquire sufficient levels of ATP 287 (combined with methionine and not using a preferred N-source) in the lung tissue to overcome 288 the growth defect resulting from targeting MetH. The concentration of free extracellular ATP in 289 human plasma has been calculated to be in the sub-micromolar range (28-64 nM) (52). In the 290 lungs, extracellular ATP concentrations must be strictly balanced and increased levels are 291 implicated in the pathophysiology of inflammatory diseases (53); nevertheless, even in such 292 cases ATP levels have been calculated in the low micromolar range (54, 55). Despite this low 293 concentrations and consequently unlikely compensation, we believe that MetH needs to be 294 validated in a suitable model to confirm that its deficiency cannot be not overcome in 295 established infections. 296 We then questioned how the lack of methionine synthase's enzymatic activity could cause a 297 drop in cell energy. We hypothesised that blockage of methionine synthase activity likely causes 298 a forced conversion of 5,10-methylene-THF to 5-methyl-THF by the action of MetF (Fig. 1A). In 299 support of this, we observed that expression of metF was increased in the H_OFF strain (Fig. 3D). 300 This likely causes a shortage of 5,10-methylene-THF, as the conversion is not reversible and THF 301 cannot be recycled by the action of methionine synthase (Fig. 1A). Indeed, supplementation of 302 folic acid (only when amino acids are the sole N-source, Fig. S3) and of purines could partially 303 restore growth ( Fig. 2A), as they compensate for the deficit in purine ring biosynthesis when 304 there is a shortage of 5,10-methylene-THF. However, this still does not explain why there is a 305 drop in ATP. We hypothesised that the block of purine biosynthesis might be sensed as a 306 shortage of nucleotides. This could then cause a shift in glucose metabolism from glycolysis and 307 the TCA cycle (which produce energy) to the Pentose Phosphate Pathway (PPP), which is 308 required to produce ribose-5-phosphate, an integral part of nucleotides. In a similar vein, it has 309 recently been described that activation of anabolism in Saccharomyces cerevisiae implies 310 increased nucleotide biosynthesis and consequently metabolic flow through the PPP (56). To 311 evaluate our hypothesis, we investigated the transcription level of the glucose-6-phosphate 312 dehydrogenase (G6PD) encoding gene (AFUA_3G08470), which catalyses the first committed 313 step of the PPP. In agreement with our hypothesis, the expression of G6PD encoding gene 314 increases in the H_OFF strain upon addition of Dox (Fig. 3D), likely reflecting an increased flow 315 through the PPP. We then wondered how cells may be activating the PPP. The target of 316 rapamycin (TOR) TORC1 effector, which is widely known to activate anabolism and growth (57-317 59), has been described to activate the PPP in mammalian cells (60, 61) and has been 318 functionally connected with energy production and nucleotide metabolism in A. fumigatus (62). 319 In addition, the cAMP/PKA (protein kinase A) pathway is known to be paramount for sensing of 320 nutrients and the correspondent adaptation of gene expression and metabolism (63), and was 321 found to be implicated in the regulation of nucleotide biosynthesis in A. fumigatus (64). 322 Consequently, we explored if a partial block of TOR with low concentrations of rapamycin or of 323 PKA with H-89 could prevent the imbalanced activation of the PPP in the absence of MetH 324 activity. However, neither of the inhibitors could reconstitute growth of the H_OFF strain in 325 restrictive conditions (Fig. S4C). This means that neither the TOR nor the PKA pathways seem to 326 be involved in the deleterious metabolic shift that seemingly activates PPP and decreases flux 327 through glycolysis. Therefore, more experiments are required to elucidate the mechanism 328 underlying the metabolic imbalance developed upon metH downregulation. 329 In summary, we propose that absence of methionine synthase activity causes a strong defect 330 in purine biosynthesis that the cell tries to compensate for by shifting carbon metabolism to the 331 PPP; this metabolic imbalance causes a drop of ATP levels, which collapses cell energetics and 332 results in halted growth (Fig. 3E). 333 Interestingly, we also detected that metF expression is higher in the H_OFF strain compared 334 to the wild-type, even in the absence of Dox (Fig. 3D). This could be explained as an effort to 335 compensate a higher demand of 5-methyl-THF by the slightly increased amount of methionine 336 synthase in this strain (Fig. S1A). This effect could cause a mild defect in purine biosynthesis in 337 the H_OFF strain, and indeed adenosine content was lower in the H_OFF-Dox condition 338 compared with the wild-type-Dox sample in the metabolome analysis ( Fig 3A). Furthermore, 339 this also explains why we detected a small but significant increase of G6PD expression in the 340 H_OFF-Dox condition (Fig. 3D). Therefore, it seems that upregulating methionine synthase has 341 the potential to cause the same metabolic imbalance as downregulating it. However, the effect 342 of overexpression (notice that it is only ~1.5 fold in our strain Fig. S1A) is minor and does not 343 have obvious consequences for growth, as THF can be recycled and thus the shortage of 5,10-344 methylene-THF is not severe. In any case, two important points can be highlighted from this 345 small imbalance. Firstly, methionine synthase activity is very important and must be finely tuned 346 to maintain a proper metabolic homeostasis. Secondly, changing the expression level of genes 347 with constitutive and/or regulatable promoters can have unexpected and hidden consequences 348 that often go unnoticed. 349

Supplementation with S-adenosylmethionine reconstitutes ATP levels and growth 351
We have shown that the absence of MetH activity causes a reduction in ATP levels. S-352 adenosylmethionine (SAM) is produced from methionine and ATP by the action of S-353 adenosylmethionine synthetase SasA (Fig. 1A), an essential enzyme in A. nidulans (65). Hence, 354 we reasoned that the absence of MetH activity might cause a decrease in SAM levels. To test 355 that hypothesis, we first attempted to rescue growth of the H_OFF strain in a medium 356 supplemented with methionine and SAM. We tested various N-sources to diversify the presence 357 of permeases in the cell membrane, aiming to maximise the chances of SAM uptake (Fig. S3). 358 Indeed, the addition of SAM reconstituted growth of the H_OFF strain in restrictive conditions 359 in the presence of methionine when amino acids were the only N-source ( Fig. 4A and S3). We 360 then measured the intracellular concentration of SAM in growing mycelia upon addition of Dox 361 using MS/MS. Surprisingly, we observed that addition of Dox to the H_OFF strain did not cause 362 a significant reduction in SAM levels (Fig. 4B). Consequently, we wondered how the addition of 363 SAM may reconstitute H_OFF growth if its levels are not reduced upon metH repression. We 364 speculated that as SAM is a crucial molecule it continues to be produced even if the levels of 365 ATP are reduced, draining it from other cellular processes and thus triggering energy 366 deprivation. In support of this hypothesis, we observed that supplementing SAM to the medium 367 increased the levels of ATP in growing hyphae ( The strain expressing wild-type MetH grew normally in restrictive conditions (Fig. S5A), proving 384 that the tagged MetH-GFP protein was active. Importantly, this result also demonstrated that 385 genetic downregulation of metH is the only reason for the lack of growth of the H_OFF strain in 386 the presence of Dox. We confirmed that A. fumigatus MetH localises in both the nucleus and 387 cytoplasm (Fig 4D & S5B). In contrast to what was described in P. pastoris, the MetH R749A protein 388 seems to be active, as it could trigger growth of H_OFF in restrictive conditions (Fig. S5A) and 389 still localised into the nucleus (Fig. S5B). Therefore, the possibility that MetH localisation in the 390 nucleus is important needs further exploration. 391 392

Repression of methionine synthase causes growth inhibition in growing mycelia 393
The major advantage of the tetOFF system is that it can be employed to simulate a drug 394 treatment before a specific chemical is developed. Addition of Dox to a growing mycelium 395 downregulates the gene of interest (Fig S1A), mimicking the effect of blocking its product by the 396 action of a drug. The validity of the Tet systems has recently been questioned, as it has been 397 reported that Dox can impair mitochondrial function in various eukaryotic models (66). Moreover, a study that investigated the role of various mitochondrial proteins for its function in 406 C. albicans did not observe any negative effect of 20 µg/mL Dox on fungal growth nor on 407 mitochondrial morphology and function (76). To test the effect of Dox on A. fumigatus, we grew 408 the wild-type strain overnight in various concentrations of drug and imaged mitochondria using 409 the Rhodamine 123 dye (Fig S6). It was previously described that inhibition of translation in 410 mitochondrial (mechanism of Dox toxicity) promotes mitochondrial fission, which can be 411 detected as a more fragmented, punctuate, mitochondrial appearance compared to the healthy 412 tubular morphology (66, 76). This fragmented phenotype started to appear, although there was 413 variation among hyphae, when the fungus was incubated in 100 µg/mL Dox and became obvious 414 when it was incubated in 1000 µg/mL Dox (Fig. S6). In contrast, in low concentrations of Dox (1  415 and 10 µg/mL Dox) the mitochondria showed a healthy tubular morphology, indistinguishable 416 from that of no Dox (Fig. S6). Therefore, low concentrations of Dox do not affect mitochondria 417 morphology and thus likely do not impair their function. In fact, we have also observed that 418 addition of 5 µg/mL Dox to wild-type mycelium did not affect the ATP content (Fig 3B), further 419 supporting the conclusion that mitochondrial function is not impaired. Therefore, even if higher 420 concentrations of Dox have a negative effect on fungal cells (70, 74), its impact at low 421 concentrations on fungal cells seems to be minimal. Consequently, we argue that as long as the 422 concentration of Dox used is ≤ 50 µg/mL, the tetOFF system can be utilised to investigate the 423 consequences of downregulating gene expression in fungal research. 424 To investigate the effect of downregulating metH for mycelial growth, we added Dox to 12, 425 16 or 24 h grown submerged mycelia and left it incubating for an additional 24 h. Addition of 426 Dox to 12 or 16 h grown mycelia severely impaired growth of the H_OFF strain but not the wt, 427 as observed by biomass (Fig. 5A) and OD (Fig. S7A) measurements. This effect was lost when 428 Dox was added to 24 h grown mycelia, due to the incapacity of Dox to reach and downregulate 429 expression in all cells within the dense mass of an overgrown mycelium. Interestingly, Dox 430 addition to methionine free media stopped H_OFF growth immediately, which can be observed 431 by comparing fungal biomass at the time of Dox addition to the measurement 24 h after Dox 432 addition. In contrast, the fungus inoculated in methionine containing media grew a little further 433 after Dox addition (Fig. 5A). To understand this difference, we added Dox to either resting or 8 434 h germinated conidia and imaged them 16 and 40 h after drug addition (Fig. 5B & Fig. S7B). In 435 agreement with the previous result, we observed that Dox addition in methionine free medium 436 inhibited growth immediately: resting conidia did not germinate and germinated conidia did not 437 elongate the germtube. In contrast, after addition of Dox in methionine containing medium, 438 most of the resting conidia were still able to germinate and some germlings could elongate the 439 germinated tubes to form short hyphae. This suggests that the drop in ATP levels takes ~3-4 h 440 before having an effect on growth. Importantly, once growth was inhibited, the effect was 441 sustained for a long period, as we could not detect further growth up to 40 h post-inoculation. 442 To corroborate these observations and further determine whether the effect of growth is 443 fungistatic or fungicidal in the long term, we performed a time-lapse analysis of the effects of 444 adding Dox to 8 h swollen conidia and its subsequent withdrawal after 16 h of incubation (Fig. 445 5C and Video 1). We observed that growth was inhibited ~4 h after Dox addition and almost 446 completely halted after 6 h, which was sustained as long as the drug was present. Upon 447 withdrawal of Dox, growth resumes within 6 h ( Fig 5D and Video S1), showing that the effect of 448 blocking MetH is fungistatic, at least with the genetic TeOFF model of metH repression. As 449 expected Dox had no effect on wild-type growth (Video S2). Consequently, these models have been used to investigate the relevance of genes required to 474 grow in vitro to initiate pulmonary infection (17,72,77). However, those models cannot be used 475 to determine the importance of the genes in established aspergillosis infections in vivo. 476 Therefore, we aimed to optimise the use of the TetOFF system for this purpose, as it can be used 477 to downregulate gene expression in growing mycelia. As a control for the model, we constructed 478 a cyp51A_tetOFFΔcyp51B (51A_OFF) strain. We reasoned that the target of the azoles, first-line 479 treatment drugs for Aspergillus diseases, should be the gold standard to compare any target 480 against. This strain showed a similar behaviour as H_OFF in vitro: as little as 0.05 µg/ml Dox 481 prevented colony development on an agar plate (Fig. S8A) and addition of Dox to conidia or 482 germlings blocked growth (Fig. S8B). 483 We first assayed the use of the TetOFF system in the Galleria mellonella alternative mini-host 484 model of infection. Preliminary experiments revealed that the balance between reaching 485 sufficient levels of Dox to exert an effect and maintaining toxic effects of overdose low was very 486 delicate. We finally optimised a regimen consisting of 5 injections of 50 mg/kg Dox (Fig. S9A) 487 that caused little mortality in the control group (25% in Dox control VS 12% in PBS treatment 488 control P=0.22) but still showed an effect of treatment (Fig. 6A). We then infected Galleria larvae 489 with 5×10 2 conidia of 51A_OFF or H_OFF strains and applied the Dox regimen or PBS vehicle 490 starting at the same time of infection (0 h) or 6 h after infection (Fig. S9A). For both strains, 491 administration of Dox from the beginning of infection triggered a significant improvement in 492 survival compared with the non-treated conditions (50% VS 17.2% for 51A_OFF, P=0.0036, and 493 41.45% VS 6.67% for H_OFF, P=0.022) (Fig. 6A). The fact that administration of Dox at the time 494 of infection did not improve survival to close to 100%, was not surprising, as it is important to 495 note that Dox does not completely prevent gene expression (Fig. S1A), so a moderate effect on 496 survival was expectable. Furthermore, rapid metabolization of the drug in the larvae hemocoel 497 or microenvironment variations in its concentration may also account for a discrete effect of 498 treatment. Despite this limitations of the model, we observed that administration of Dox 6 h 499 after infection also triggered a significant improvement in survival for both strains (42.8% VS 500 17.2% for 51A_OFF, P=0.0007, and 32.26% VS 6.67% for H_OFF, P=0.0324) (Fig. 6A). Therefore, 501 downregulation of methionine synthase genetic expression in established infections conferred 502 a significant benefit in survival which was comparable to that observed with the target of the 503 azoles. 504 The positive results obtained using the Galleria infection model prompted us to assay the 505 TetOFF system in a leukopenic murine model of pulmonary aspergillosis. To ensure that Dox 506 levels in mouse lungs reach and maintain sufficient concentrations to downregulate gene 507 expression (according to our results in vitro) we performed a pilot Dox dosage experiment in 508 immunosuppressed non-infected mice (Fig S8B). We extracted lungs of Dox treated mice at 509 different time-points, homogenated them and measured Dox concentration using a bioassay 510 based on inhibition of Escherichia coli DH5α growth. We could detect promising levels of Dox in 511 all mice (concentrations ranging from 2.2 to 0.94 µg/mL - Fig. S9B-) which according to our 512 results in vitro should be sufficient to downregulate gene expression from the TetOFF system. 513 We therefore infected leukopenic mice with 10 5 spores of the 51A_OFF or the H_OFF strains and 514 administered PBS vehicle or our Dox regimen, starting 16 h after infection (Fig. S9B). The use of 515 an uninfected, Dox treated control group uncovered that the intense Dox regimen used was 516 harmful for the mice. These uninfected mice lost weight at a similar rate as the infected groups 517 and looked ill from the third or fourth day of treatment. This is not surprising as Dox can impair 518 mitochondrial function in mice (66) and has iron chelating properties (78). As a consequence, 519 there was no beneficial effect of Dox treatment on survival (not shown). The fact that Dox 520 treatment did also not show any benefit in survival for our control strain 51A_OFF, which should 521 mimic treatment with azoles (primary therapy for invasive aspergillosis), indicates that the 522 TetOFF system is not ideal to mimic a drug treatment in established infections. Nevertheless, we 523 further attempted to determine the efficiency of targeting MetH in established infections by 524 measuring fungal burdens in lungs of treated and untreated mice. We observed that two and a 525 half days of Dox treatment (when the mice have not developed visible toxic effects yet) did result 526 in a significant reduction of fungal burdens 3 days after infection for both 51A_OFF (P=0.0279) 527 and H_OFF (P=0.0019) (Fig. 6B). Therefore, we could observe a beneficial effect of interfering 528 with methionine synthase genetic expression in an established pulmonary infection, which was 529 comparable to that of interfering with the expression of cyp51A, the target of azoles. This 530 constitutes a very rigorous validation of MetH as a promising antifungal target. 531 A recent study also aimed to use another TetOFF system to validate a drug target in 532 established aspergillosis infections (79). These authors administered Dox exclusively through 533 oral gavage, accounting for lower dosage of drug. Consequently, even if no toxic effect for the 534 mice was observed, they also did not detect any beneficial effect on survival when the Dox 535 treatment was initiated after infection. Therefore, the TetOFF system is clearly not optimal and 536 better models are needed. Yet, it is currently the only model with which the efficiency of new 537 targets can be tested in aspergillosis established infections, and thus it is highly valuable that we 538 have been able to optimise its use in vivo. Our 51A_OFF control strain has been key to calibrating 539 the model, and allows us to be confident that the beneficial effects observed, even if subtle, are 540 significant. Hence, we propose that hereinafter proper genetic validation of antifungal targets 541 should include testing their relevance in established infections. 542 543

Structural-based virtual screening of MetH 544
Having shown in vivo that MetH is a promising target, we decided to investigate its druggability 545 by running a structural-based virtual screening. The sequence of A. fumigatus MetH (AfMetH) 546 contains two predicted methionine synthase domains with a β-barrel fold conserved in other 547 fungal and bacterial enzymes. The structure of the C. albicans orthologue (80) (CaMetH) showed 548 that the active site is located between the two domains where the methyl tetrahydrofolate, the 549 homocysteine substrate and the catalytic zinc ion bind in close proximity. The homology model 550 for AfMetH (Fig. 7A) overlaps very well with that of the CaMetH thus providing a suitable 551 molecular model for further analysis. In contrast, the structure of the human methionine 552 synthase (hMS) shows a very different overall arrangement with the folate and homocysteine 553 binding domains located in completely different regions (Fig. 7B). Comparison of the 554 tetrahydrofolate binding sites between the fungal and the human structures also highlights 555 significant structural differences that affect the conformation adopted by the ligand. In the 556 CaMetH structure the 5-methyl-tetrahydrofolate (C2F) adopts a bent conformation (<20Å long) 557 and it is in close proximity to the methionine product, whereas in the human structure the 558 tetrahydrofolate (THF) ligand binds in an elongated conformation extending up to 30Å from end 559 to end, (Fig. 7 C&D). 560

Virtual screening (VS) was carried on the AfMetH and the hMS structures with the Maybridge 561
Ro3 fragment library to explore potential venues for drug development. The results showed four 562 ligand binding clusters in the AfMetH structure, two of which (C1, C2) match the binding position 563 of the 5-methyl-tetrahydrafolate and the methionine from the CaMetH crystal structure (Fig.  564   7E). For the hMS, we found two main clusters, C1 that overlaps with the tetrahydrofolate binding 565 site and C2 in a nearby pocket. Clearly the distribution of the clusters defines a very different 566 landscape around the folate site between the human and the fungal enzymes. Furthermore, the 567 proximity of the C1 and C2 clusters, matching the folate and Met/homocysteine binding sites in 568 the Ca/Af proteins means that it may be possible to combine ligands at both sites to generate 569 double-site inhibitors with high specificity towards the fungal enzymes. Antifolates are a class of 570 drugs that antagonise folate, blocking the action of folate dependent enzymes such as 571 dihydrofolate reductase (DHFR), thymidylate synthase or methionine synthase. Methotrexate is 572 an antifolate commonly used to treat cancer and autoimmune diseases. Interestingly, 573 methotrexate has been shown to be a weak inhibitor of the C. albicans methionine synthase 574 (32) and to have some antifungal activity against C. albicans (81) and Aspergillus ssp (82). 575 Nevertheless, methotrexate is not a good antifungal drug, as its activity is high against human 576 enzymes (IC50 of 0.3 µM for DHFR (83)) and low against fungal methionine synthase (IC50 of 4 577 mM for C. albicans MetH (32)). Therefore, more potent and specific inhibitors of fungal 578 methionine synthases are needed to fully exploit the value of this target for antifungal therapy, 579 a task that seems possible and can be directed from our analyses. 580 581 In summary, we have shown that methionine synthase blockage triggers not only methionine 582 auxotrophy, but also a metabolic imbalance that results in a drop in cellular energetics and 583 growth arrest. In light of our results, we stress that conditional essentiality is important to 584 understand the underlying mechanisms of metabolic processes and needs to be considered to 585 achieve proper validation of novel antimicrobial targets. Accordingly, we proved that targeting 586 methionine synthase in established infections has a beneficial effect similar to that observed for 587 the target of azoles, the most effective drugs for the treatment of aspergillosis. Finally, we 588 showed that fungal methionine synthases have distinct druggable pockets that can be exploited 589 to design specific inhibitors. In conclusion, we have demonstrated that fungal methionine 590 synthases are promising targets for the development of novel antifungals. 591 592 593

MATERIAL AND METHODS 594
Strains, media and culture conditions 595 The Escherichia coli strain DH5α (84) was used for cloning procedures. Plasmid-carrying E. coli 596 strains were routinely grown at 37°C in LB liquid medium (Oxoid) under selective conditions (100 597 µg•mL -1 ampicillin or 50 µg•mL -1 kanamycin); for growth on plates, 1.5% agar was added to 598 solidify the medium. All plasmids used in the course of this study were generated using the 599 Seamless Cloning (Invitrogen) technology as previously described (17,85). E. coli strain BL21 600 (DE3) (86) was grown on Mueller Hinton agar (Sigma) in bioassays, to determine Dox 601 concentrations within homogenized murine lungs. 602 The wild-type clinical isolate Aspergillus fumigatus strain ATCC 46645 served as reference 603 recipient. A. fumigatus strain A1160 (ku80Δ) (87) was also used to confirm metH essentiality. A. 604 fumigatus mutants were generated using a standard protoplasting protocol (88) III was used, with a Q-imaging Retinga 6000 camera, and manipulated using Metamorph v7760. 630 Confocal imaging was performed using a Leica TCS SP8x inverted confocal microscope equipped 631 with a 40X/0.85 objective. Nuclei were stained with DAPI (Life Technologies Ltd) as described 632 previously (94). GFP was excited at 458 nm with an Argon laser at 20% power. DAPI was excited 633 at 405 nm with an LED diode at 20%. 634

Metabolome analyses 635
A. fumigatus wild-type and metH_tetOFF strains were incubated in MM for 16 h before the -Dox 636 samples were taken (8 replicates of 11 mL each). Then, 5 µg/mL Dox and 5 mM methionine (to 637 prevent metabolic adaptation due to met auxotrophy) were added as appropriate and the 638 cultures incubated for 6 h, after which the +Dox samples were taken (8 × 11 mL). The samples 639 were immediately quenched with 2× volumes of 60% methanol at -48°C. After centrifugation at 640 4800 g for 10 min at -8°C, metabolites were extracted in 1 mL 80% methanol at -48°C by three 641 cycles of N2 liquid snap freezing, thawing and vortexing. Supernatant was cleared by 642 centrifugation at -9 °C, 14,500 g for 5 min. Quality control (QC) samples were prepared by 643 combining 100 µL from each sample. Samples were aliquoted (300 µL), followed by the addition 644 of 100 µL of the internal standard solution (0.2 mg/mL succinic-d4 acid, and 0.2 mg/mL glycine-645 For data analysis, the GC-MS raw files were converted to mzXML and subsequently imported 661 to R. The R package "erah" was employed to de-convolve the GC-MS files. Chromatographic 662 peaks and mass spectra were cross-referenced with the Golm library for putative identification 663 purposes, and followed the metabolomics standards initiative (MSI) guidelines for metabolite 664 identification (96). The peak intensities were normalised according to the IS (succinic-d4 acid) 665 before being log10-scaled for further statistical analysis. All pre-processed data were investigated 666 by employing principal component analysis (PCA) (97). 667 The raw data of this metabolome analysis has been deposited in the MetaboLights database 668 (98), under the reference MTBLS1636 (www.ebi.ac.uk/metabolights/MTBLS1636) 669

ATP Quantitation 670
A. fumigatus was grown as in the metabolome analysis. However, where the effect of SAM was 671 investigated spores were inoculated into MM-N + 1mg/mL aac and 0.5mM SAM was also added 672 at the time of Dox addition. ATP levels were determined using the BacTiter-Glo TM Assay 673 (Promega) following the manufacturer's instructions and a TriStar LB 941 Microplate Reader 674 (Berthold). 675

Isolation and detection of SAM 676
A. fumigatus was grown exactly in the same conditions as described for the metabolome 677 analysis. Harvested mycelia were snap-frozen in liquid N2 and stored at -70 °C before SAM 678 isolation. SAM extraction was carried out according to Owens et al (99). Briefly, frozen mycelia 679 were ground in liquid N2 and 0.1 M HCl (250 µL) was added to ground mycelia (100 mg). Samples 680 were stored on ice for 1 h, with sample vortexing at regular intervals. Samples were centrifuged 681 at 13,000 g for 10 min (4 °C) to remove cell debris and supernatants were collected. 682 Concentration of protein in supernatants was determined using a Biorad Bradford protein assay 683 relative to a bovine serum albumin (BSA) standard curve. Clarified supernatants were adjusted 684 to 15 % (w/v) trichloroacetic acid to remove protein. After 20 min incubation on ice, 685 centrifugation was repeated and clarified supernatants were diluted with 0.1 % (v/v) formic acid. 686 Samples were injected onto a Hypersil Gold aQ C18 column with polar endcapping on a Dionex 687 UltiMate 3000 nanoRSLC with a Thermo Q-Exactive mass spectrometer. Samples were loaded in 688 100 % Solvent A (0.1 % (v/v) formic acid in water) followed by a gradient to 20 % B (Solvent B: 689 0.1 % (v/v) formic acid in acetonitrile) over 4 min. Resolution set to 70000 for MS, with MS/MS 690 scans collected using a Top3 method. SAM standard (Sigma) was used to determine retention 691 time and to confirm MS/MS fragmentation pattern for identification. Extracted ion 692 chromatograms were generated at m/z 399-400 and the peak area of SAM was measured. 693 Measurements were taken from three biological and two technical replicates per sample, 694 normalized to the protein concentration in the extracts from each replicate. SAM levels are 695 expressed as a percentage relative to the parental strain in the absence of Dox. 696

Nuclei isolation and Western Blot 697
Protoplasts were generated as in A. fumigatus transformations and nuclei isolated were isolated 698 by sucrose gradient fractionation as previously described by Sperling and Grunstein (100). 699 Nuclear localisation of GFP-tagged target proteins was confirmed by Western-blot. Aliquots of 700 nuclei were boiled for 5 minutes in loading buffer (

Biomass measurement 719
Conidia were inoculated into MM-S, supplemented with either methionine or sulfate, and 720 incubated at 37°C 180 rpm for 12, 16 or 24 h. After this initial incubation, 3 mL samples were 721 taken in triplicate from the cultures, filtered through tared Miracloth, dried at 60°C for 16 h and 722 their biomass measured. In treated conditions Dox was added to a final concentration of 1 723 μg/mL and the culture allowed to grow for a further 24 h at 37°C 180 rpm. 5 mL samples were 724 taken in triplicate and their biomass measured as above. 725

Galleria mellonella infections 726
Sixth-stage instar larval G. mellonella moths (15 to 25 mm in length) were ordered from the Live 727 Foods Company (Sheffield, United Kingdom). Infections were performed according to Kavanagh 728 and Fallon (103). Randomly selected groups of 15 larvae were injected in the last left proleg with 729 10 µL of a suspension of 5×10 4 conidia/mL in PBS, using Braun Omnican 50-U 100 0.5-mL insulin 730 syringes with integrated needles. Dox was administered according to the treatment shown in 731 Fig. S9A, alternating injections in the last right and left prolegs. In each experiment an untouched 732 and a saline injected control were included, to verify that mortality was not due to the health 733 status of the larvae or the injection method. Three independent experiments were carried out. 734 The presented survival curves display the pooled data, which was analysed with the Log-Rank 735 test. 736 burden, 500 ng of DNA extracted from each infected lung were subjected to qPCR. Primers used 757 to amplify the A. fumigatus β-tubulin gene (AFUA_7G00250) were forward, 5'-758 ACTTCCGCAATGGACGTTAC-3', and reverse, 5'-GGATGTTGTTGGGAATCCAC-3'. Those designed 759 to amplify the murine actin locus (NM_007393) were forward, 5'-CGAGCACAGCTTCTTTGCAG-3' 760 and reverse, 5'-CCCATGGTGTCCGTTCTGA-3'. Standard curves were calculated using different 761 concentrations of fungal and murine gDNA pure template. Negative controls containing no 762 template DNA were subjected to the same procedure to exclude or detect any possible 763 contamination. Three technical replicates were prepared for each lung sample. qPCRs were 764 performed using the 7500 Fast Real-Time PCR system (Thermo Fisher Scientific) with the 765 following thermal cycling parameters: 94 °C for 2 min and 40 cycles of 94°C for 15 s and 59ᵒC for 766 1 min. The fungal burden was calculated by normalising the number of fungal genome 767 equivalents (i.e. number of copies of the tubulin gene) to the murine genome equivalents in the 768 sample (i.e number of copies of the actin gene) (104). Two independent experiments were 769 carried out (n=9, 5 mice in the first and 4 mice in the second experiment). Burdens for each 770 strain were compared using a Mann Whitney test. 771

Molecular homology models and virtual screening 772
The full-length sequence for AFUA_4G07360, the cobalamin-independent methionine synthase 773  (3)

COMPETING INTERESTS 809
The authors declare no competing interests. 810

DATA AVAILABILITY 811
The raw data that support the findings of this study are available upon reasonable request to 812 the authors. The raw data of metabolome analysis has been deposited in the MetaboLights 813   (1) D616A (2)