Functional, transcriptomic, and lipidomic studies of the choC gene encoding a phospholipid methyltransferase in Aspergillus fumigatus

ABSTRACT Phosphatidylcholines (PCs) are a class of cell membrane phospholipids playing important roles in cellular integrity and signaling. In eukaryotes, PCs are biosynthesized via the Kennedy cytidine nucleotide and the Bremer-Greenberg pathways. The Aspergillus choC gene is predicted to encode a phospholipid methyltransferase, the key enzyme in the latter pathway. In this study, we have characterized choC in the opportunistic human pathogenic fungus Aspergillus fumigatus and show that ChoC plays a vital role in the endogenous biosynthesis of PC, which is necessary for proper vegetative growth, development, cell viability, and cell wall integrity of the fungus. Transcriptomic analyses have revealed that the deletion (Δ) of choC leads to the differential expression of diverse genes related to PC biosynthetic pathways, G protein signaling pathways, cell cycle, and autophagy. Importantly, the ΔchoC mutant averted cell death and regained cellular proliferation by regulating the expression of different PC synthesis genes after 4 days of incubation. Lipidomic analyses have demonstrated that ΔchoC directly leads to decreased PC synthesis, thereby affecting the length and unsaturation of PC fatty acid chains. In addition, ΔchoC leads to disturbed metabolism of other lipids, such as reduced phosphatidylethanolamine and neutral lipid triacylglycerol, but increased diacylglycerol amounts. Murine infection studies showed that invasive pathogenicity of the ΔchoC spores was significantly weakened coupled with a drastic reduction of their ability to colonize in kidneys. In summary, ChoC plays central roles in PC biosynthesis, lipid metabolism, cell membrane/wall integrity, endoplasmic reticulum function, and pathogenicity in A. fumigatus, implying that ChoC can be a novel target for antifungal therapy. IMPORTANCE This study explored the phospholipid metabolic pathway in A. fumigatus and its relationship with fungal growth, metabolism, and pathogenicity. ChoC, based on its critical roles in many aspects of the fungus and relatively conserved characteristics in filamentous fungi with low similarity with mammalian ones, can be a novel target of new antifungal drugs.

re-introducing the choC wild-type (WT) allele into the deletion mutant separately.As shown in Fig. 1D, the ΔchoC mutant exhibited severely impaired vegetative growth and lacked asexual development on day 3 in minimal medium (MM).The addition of diethanolamine (DAE) or choline, but not serine, ethanolamine (EA), or monoethanol amine (MAE), completely restored vegetative growth of the ΔchoC mutant (Fig. 1D).However, when DAE or choline was added, the ΔchoC mutant displayed near-complete restoration of conidiation (Fig. 1E).These results indicate that the functions of choC are conserved in A. fumigatus and are involved in the last two steps of the Bremer-Greenberg methylation pathway, from phosphoryl monoethanolamine to phosphoryl diethanola mine (PDAE) and PDAE to PC. PC is essential for vegetative growth and development of Aspergillus species.

ChoC is necessary for proper proliferation, cell viability, and cell wall integrity in A. fumigatus
To further determine the role of choC in cell proliferation, we examined the mycelial dry weight of the WT (A. fumigatus 293.1) and ΔchoC strains in liquid MM, MM + DAE, and MM + choline.As shown in Fig. 2A, the mycelial dry weight of the WT strain reached a maximum on day 2 in MM, while the dry weight of ΔchoC strains reached a maximum on day 3 and showed severely restricted vegetative growth during the first 2 days of incubation.After the addition of DEA and choline, the vegetative growth of ΔchoC was almost restored.Moreover, the incubation of the ΔchoC mutants in liquid MM led to the formation of large mycelial aggregates with protruding hyphae on day 6 (Fig. 2B).To evaluate the role of choC in cell viability (measured by mitochondrial activity), we determined the reduction rates of alamar blue from WT and ΔchoC strains grown in liquid MM, MM + DAE, and MM + choline medium.WT strains reached a peak in cell viability on day 3 and then a slow decline in cell viability was observed from day 3 to day 6 in MM, while the ΔchoC mutant cells displayed high cell viability on day 2, but a sharp reduction in cell viability from day 3 to day 6.After adding DAE and choline, the ΔchoC mutant cells regained viability, which was comparable to that of WT (Fig. 2C).We also determined cell death (apoptosis) levels in WT and ΔchoC strains using Evans blue staining.As shown in Fig. 2D, compared to WT, ΔchoC strains displayed precocious cell death on day 2 in MM, while on day 6 ΔchoC strains could escape cell death and regenerate new viable hyphae from big mycelial aggregates.To test whether the absence of choC affected the integrity of the cell wall, we investigated the hyphal morphology of the ΔchoC mutant cells in MM containing 1.2 M sorbitol and found that the swollen hyphae of the ΔchoC mutant were partly restored by osmotic stabilizers and normal hyphae were generated (Fig. 2E).When 250 mg/L Congo red was added to MM, the ΔchoC mutant grew less well than WT and more swollen structures appeared at the tips of the mutant hyphae (Fig. 2F), suggesting that hyphal tip swelling was likely caused by defective cell walls.These results indicate that choC deficiency affects cell proliferation, viability, death, and wall integrity in A. fumigatus and that the function of choC is conserved in A. fumigatus.

Genome-wide expression analyses of the ΔchoC mutant
The choC gene encodes an important methylation enzyme essential for A. fumigatus to synthesize PC.To explore the genes and gene networks involved in cell proliferation, cell death, and cell reprogramming with the disturbance of PC homeostasis, we performed RNA-Seq analyses of WT and the ΔchoC mutant on days 2 and 4 of cultivation in MM (Table S1).

Disruption of PC synthesis has a negative impact on energy metabolism, material transformation, and signal regulation in ΔchoC
To determine the DEGs involved in the major biochemical, metabolic, and signal transduction pathways, KOBAS software was used to test the statistical enrichment of DEGs in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (Fig. 4A through D) (42,43).Genes with significant differences in the expression of KEGG pathways among the four comparative combinations are shown in Tables S5 to S8 (log 2 FC > 0.5, P < 0.05).On day 2, the significantly upregulated DEGs in the ΔchoC cells were mainly involved in primary metabolic pathways related to energy metabolism, such as oxidative phosphorylation and the pentose phosphate pathway; the biosynthesis pathway of amino acids such as valine, linoleic acid, and butyric acid; and other pathways related to lipid metabolism and the synthesis of secondary metabolites.The downregula ted genes mainly focused on the biosynthesis of amino acids, the assembly of ribosomes, and other pathways related to protein metabolism, the metabolism of purine, pyrimi dine, and RNA, and the biosynthesis of fatty acids.On day 4, the significantly upregulated DEGs were mainly involved in lipid metabolism, such as the biosynthesis and metabolism of unsaturated fatty acids and other fatty acids; the metabolic pathways of sphingolipids, steroids, and glycerides; the occurrence of proteins in the endoplasmic reticulum (ER); the metabolic pathways related to glucan synthesis; and the signal metabolic pathways, such as inositol phosphate and MAPK signaling pathways.It was noticeable that CK1, encoding choline kinase, in the ΔchoC mutant practically had no mRNA levels compared to WT on days 2 and 4, as shown in Tables S5 and S6.According to the results of the KEGG experiment, we hypothesized that CK genes may be closely related to choC and knocked out two genes, CK1 and CK2, in WT and the ΔchoC mutant, respectively.Growth of ΔCK1 and ΔCK2 strains was slightly reduced compared to that of WT strains, indicating that the deletion of CK1 or CK2 in WT has a minor impact on A. fumigatus.However, growth ΔCK1 and ΔCK2 was significantly better than that of the ΔchoC mutant on solid MM.The ΔchoCΔCK1 double mutant grew poorly on MM even with the choline supplementation or on minimal medium with yeast extract (MMY).This result indicates that the absence of CK1 and ChoC functions has a synergistic adverse effect on A. fumigatus growth (Fig. S3).

Lipidomic analyses of choC function in A. fumigatus
The synthesis and catabolism of different types of lipids cross each other, particularly in lipid synthesis.PE is an intermediate metabolite of the methylation pathway that synthesizes PC and is closely related to the two main pathways of PC synthesis.Knockout of choC not only affects the synthesis of PC but also interferes with the balance of PE metabolism.To assess the effect of ΔchoC on the lipid profile of A. fumigatus, we systematically analyzed the lipidomes of ΔchoC strains compared to that of WT strains cultured for 2 and 4 days in MM, respectively.Detailed information for each group is listed in Table S2.We harvested the mycelium of WT strains and ΔchoC strains cultivated in liquid MM on days 2 and 4. The results showed that the PC content in the WT strain was significantly reduced on day 4 compared to that on day 2, and most of the reduced PCs produced fatty acyl chains with 32 and 34 carbons (Fig. S4A).The ΔchoC mutant showed the opposite results.The PC contents of the mutant were much higher after culturing for 4 days than after 2 days (Fig. S4B).The PC content of WT strain was higher The metabolic pathways of significantly downregulated DEGs in the ΔchoC mutant at 4 days.Rich factor refers to the ratio of the number of differentially expressed genes enriched in the pathway to the number of annotated genes.The greater the "Rich factor, " the greater is the degree of enrichment; q-value is the P-value after multiple hypothesis test corrections.The q-value ranges within (0, 1), and the closer to 0, the more significant the enrichment is.The first 20 entries that show the most significant enrichment are shown.
than that of the ΔchoC mutant (Fig. 5A).On day 2, the content of PEs (28:2, 29:0, 29:1, 33:2, 33:3, 33:4, 34:1, 34:2, and 34:3) in the ΔchoC mutant was lower than that in WT strain (Fig. 5B).On day 4, the PE content in the mutant generally decreased compared to that in WT, except for the PE molecule (38:4) (Fig. 5C).These results indicate that the loss of choC disturbs PE anabolism and catabolism.Although the expression of many genes in the PE metabolic pathway was upregulated, the content of PEs in the ΔchoC mutant was significantly reduced.The abundance of intracellular neutral lipids was also significantly affected in the ΔchoC mutant.The TAG content in the ΔchoC mutant was significantly lower than that in WT on day 4, except for the TAG molecules (59:6 and 54:6) (Fig. 5D).The abundance of DAG, which comes from the breakdown of TAG, in the ΔchoC mutant remarkably increased compared to that in WT at both time points (Fig. 5E).Combined with changes in PCs and PEs, ΔchoC led to the inhibition of phospholipid biosynthesis.It is necessary to mobilize neutral lipids stored in cells.As the decomposition of TAG can provide fatty acyl chains for the synthesis of lipids, the TAG content in the ΔchoC mutant decreased, and accordingly, the content of DAG, the degradation product of TAG, increased significantly.

ChoC is required for the pathogenesis of A. fumigatus
Since the viability of the ΔchoC mutant was much lower than that of WT with early onset of apoptosis, we examined the effects of choC on the pathogenesis of A. fumigatus.Mice were infected with 3 × 10 6 conidia of WT, the ΔchoC mutant, and complemented strains by tail vein injection and observed daily.The survival curve of the mice showed that almost all mice survived on day 5, and most died between days 5 and 10.The virulence of mutant strain was much lower than that of WT strain, and the invasive lethality in mice was significantly reduced (Fig. 6A).Simultaneously, we examined the fungal burden in the liver (Fig. 6B), spleen (Fig. 6C), and kidneys (Fig. 6D) of mice at 3, 10, and 17 days after infection.After the spores entered the body, the immune system of the mice responded quickly, and the spores in the liver and spleen were cleared within 3-10 days.Over time, these spores gradually transferred to and colonized the kidney.The number of spores in the kidney gradually increased in mice infected with conidia of WT and complemented strains.For the mutant strains, the number of conidia entering the kidney was higher than that of the WT and complementation strains at day 3, but decreased significantly and was lower than that of the WT strains at days 10 and 17.These indicate that the deletion of choC reduced the ability of A. fumigatus conidia to colonize the kidney, making it more likely to be cleared by immune cells in the blood.
Histopathological examination of the kidneys obtained from mice infected with WT, mutant, and complemented strains was performed at three selected time points: 3, 13, and 23 days.At day 23, periodic acid-Schiff (PAS) and hematoxylin-eosin (HE) staining showed that the kidney tissues infected with WT strains showed severe damage after the invasion of spores and a swollen disrupted glomerulus (Fig. 6E).Significant inflammatory cell infiltration was observed in the kidneys of mice infected with WT and complemented strains, indicating a strong immune response caused by immune cells phagocytizing the spores.However, the ΔchoC conidia did not induce a significant immune response in host cells.This indicates that the loss of choC reduces the virulence of A. fumigatus spores.

DISCUSSION
Our comprehensive functional studies have revealed that choC plays a pivotal role in cell viability, asexual reproduction, and cell death in A. fumigatus.These results are consistent with our previous studies done in the model fungus Aspergillus nidulans (26).Further more, our multi-omics studies have elucidated the molecular mechanism by which ChoC controls the synthesis of PCs and regulates the growth and metabolism of A. fumigatus.
The MCK1-Cdc6 pathway is recognized in yeast cells to prevent DNA replication in time after damage to the plasma membrane, which ensures that DNA replication occurs only when cell wall integrity is guaranteed (44).Insufficient PC synthesis has a great influence on the vegetative growth of A. fumigatus because of the vital role of PC in the membrane structure.Loss of choC directly led to the reduction of PC biosynthesis in A. fumigatus, and the PC content in the ΔchoC mutant was significantly lower than that of WT, but only in 2 days rather than 4 days according to our study.The colony diameter of the ΔchoC mutant on solid MM was much smaller than that of WT because the extension of hyphae requires a large amount of PC.The lack of choC interrupted the CDP-DAG pathway for intracellular PC synthesis, and the supply of PC was greatly blocked, resulting in a weakening of the growth dynamics of the hyphae (45).
Through metabolomics analyses, when cultivated in the MM for 2 and 4 days, the deletion of choC completely inhibited the expression of the choline kinase CK1 in the CDP-choline pathway, indicating that in A. fumigatus, the two PC synthesis pathways were closely related (46).When both the CK1 and CK2 genes were knocked out, it did not significantly impact the growth and development of A. fumigatus as severe as the choC deletion.It is possible that choline kinase is not a rate-limiting enzyme in the CDPcholine pathway and that there are two isoenzymes, CK1 and CK2, which are responsible for the phosphorylation of choline in A. fumigatus.Their individual deletions did not affect fungal cells as severe as the lack of choC.We did not obtain double-gene deletion strains for choC and CK2 after several attempts.It was suspected that the CK2 gene might play an indispensable role in the growth of A. fumigatus in the absence of choC.
The ER is the leading site of phospholipid synthesis in cells (47).When choC is knocked out, inhibition of PC synthesis causes an imbalance in lipid levels.According to the transcriptome results of mutant strains cultured in MM on day 2, we found that the imbalance in PC synthesis caused by the deletion of choC gene induced the ER stress response, and mRNA levels of genes involved in protein synthesis, secretion, transmission between membrane vesicles, protein lysinization, and degradation in the ER changed significantly (48).Almost all proteins of ribosomal subunits, some genes related to the protein-directed transport between the endoplasmic reticulum and Golgi apparatus, and the endoplasmic reticulum and mitochondria, as well as membrane vesicles, also showed a significant upward trend (49).These data suggest that the synthesis and catabolism of proteins in cells are accelerated by ΔchoC, which further leads to ER stress and triggers the unfolded protein response (UPR) (50).If endoplasmic reticulum stress is not properly resolved, UPR leads to cell death (51,52).
In eukaryotes, the glycosylphosphatidylinositol (GPI) anchor is a relatively conserved structure that is an important post-translational modification of many cell surface proteins (such as enzymes, receptors, and adhesion factors), transferring these proteins from their synthesis sites to their final residence (36).Bruneau et al. identified nine GPI anchor proteins in A. fumigatus, of which five (Csa1p, Crh1p, Crh2p, Ecm33p, and Gas1p) had homologs in yeast cells (53).Genetic experiments have shown that these proteins play important roles in the morphogenesis of cell wall (54).The addition of Congo red blood cell sensitizer and transcriptome results confirmed that the normal synthesis of the GPI anchor would be affected when PC synthesis was disordered.The GPI anchor was closely connected to the cell wall, and its abnormality would undoubtedly affect the integrity of the cell wall.In addition, the inhibition of GPI anchor synthesis in A. fumigatus leads to abnormal mycelial growth and asexual development (36).It has been found that the virulence of gene-defective strains in immune-deficient mice is also reduced by co-infection with normal and gene-defective strains (55).For pathogenic fungi, the cell wall is crucial for their virulence and pathogenicity, which not only endows fungal cells with the ability to adhere to host tissues but also protects cells from the influence of the host defense system (56).Therefore, the absence of choC negatively impacts the growth, reproduction, and cytotoxicity of A. fumigatus.
We found that the lack of choC affected cell growth.However, there is a precise and delicate strategy for the ΔchoC mutant to exert limited resources under the conditions of nutrient insufficiency.Lipid droplets (LDs) store excess fatty acids, such as TAG and steryl esters (STE), to prevent them from poisoning cells.These two neutral lipids can be synthesized by transferring acyl groups from fatty acids or phospholipids to DAG or sterols.In eukaryotic cells, excess fatty acids are stored in lipid droplets.Autoph agy is triggered when the cells are in a state of starvation.Lysosomes and LDs have the function of converting TAG and STE into fatty acids and cholesterol to provide the nutrients needed for growth and to maintain the balance of lipids (57).Loss of the choC gene disrupts the CDP-DAG pathway for PC synthesis in cells, resulting in impaired synthesis of PC.In the glycerolipid metabolism pathways, mRNA levels of the gene encoding TAG lipase increased 1.0 time, which was in parallel with the lipidomic results that the TAG content in the ΔchoC mutant was significantly reduced at day 2 compared with WT, and DAG levels increased accordingly.DAG is not only an important intermediate of the phospholipid metabolic network but also an intracellular secondary messenger molecule that is involved in the phosphatidylinositol signaling pathway, MAPK signaling pathway, etc., regulating physiological activities to cope with pressure inside or outside the cell (58)(59)(60).We believe that the ΔchoC mutant uses this mechanism of lipid autophagy to maintain intracellular lipid homeostasis and survive.
In fungi, the CDP-DAG and CDP-choline pathways cooperate to ensure PC biosyn thesis in fungal cells.By knocking out choC, we completely eliminated the CDP-DAG pathway and disrupted the homeostasis of PC metabolism, thus triggering various cellular changes.After finding abnormal conditions, the cell's first response is to correct them immediately.The biosynthesis of PC in the ΔchoC mutant was restored through the CDP-choline pathway with the absorption of exogenous choline, and the mutant almost completely regained the normal growth levels of WT when choline was added to MM (61).Although there is no choline supplement in MM, A. fumigatus can use limited resources to increase PC synthesis.According to the transcriptomic results, in the glycerophospholipid metabolism pathway (Fig. S5), mRNA levels of the genes encoding the PS decarboxylase (4.1.1.65)increased 4.8 times, while the lipidomic results showed that the content of PE in the ΔchoC mutant after 2 days of cultivation decreased.This may be because the degradation rate was much higher than the generation rate.Transcript levels of genes encoding phospholipase A2 PlaA (3.1.1.4),which degrades PE to LPE, and lysophospholipase Plb3 (3.1.1.5),which transfers LPE to glycerol-3-phos phoethanolamine, increased and the gene (2.7.1.82)responsible for the phosphorylation of ethanolamine increased 1.8 times.All the data display another pathway (as shown in Fig. S5 with the red curve) for PC synthesis other than the two known pathways.Phosphoethanolamine is added to the three methyl groups to generate phosphocholine, thus entering the CDP-choline pathway to synthesize PC.Based on our results, we hypothesize that other methylation enzymes in cells may play an important role in the methylation reaction for the PC synthesis in the absence of ChoC.
In summary, ChoC directly affects PC synthesis, thereby interfering with the metabolism of other lipids, such as PE, TAG, and DAG, damaging cell wall integrity, and weakening the pathogenicity in A. fumigatus.This study explored the phospholipid metabolic pathway in A. fumigatus and its relationship with growth and metabolism.ChoC, based on its relatively conserved characteristics in filamentous fungi and its special function for mammals, may be an excellent target for new antifungal drugs.

Strains, growth conditions, and media
The strains used in this study are listed in Table 1. A. fumigatus 293.1 was the original WT strain used in the present study.Minimal medium was used as the basic medium, and supplements were added according to our experimental design.MM was supplemented with 0.1% yeast extract to harvest A. fumigatus spores.To explore the function of choC in the methylation pathway, 50 mg/L L-serine, 40 mg/L ethanolamine, 50 mg/L N-methyla minoethanol, 60 mg/L N, N-dimethylaminoethanol or 50 mg/ L choline chloride, 1.2 M sorbitol, and 2.5 mg/mL Congo red were added to the MM.For liquid-submerged culture, 1 × 10 6 conidia/mL were inoculated into 100 mL liquid media and cultured at 37°C and 120 rpm.All strains were cultivated at 37°C.For the induction of asexual development, mycelia cultured in liquid MM medium at 37°C for 18 h were transferred to solid plates and cultured at 37°C as described (62).Photomicrographs were captured using an Eclipse 80i epifluorescence microscope (Nikon, Tokyo, Japan).Culture plates and northern blot images were obtained using a DSC-F707 digital camera (Sony, Tokyo, Japan).

Determination of the choC ORF
Total RNA was extracted from mycelia cultured in liquid-merged medium for 48 h and reverse transcribed into cDNA (Thermo Fisher Scientific Inc., USA).Random primers for oligo-dT and cyclic reverse transcriptase were used in the reaction.To amplify cDNA through PCR reactions, we used primers c-choC S (upstream of the 5′ end of the choC ORF) and c-choC AS (downstream of the 3′ end of the choC ORF).Genomic DNA was amplified using choC S (upstream of the 5′ end of the choC ORF) and choC AS (down stream of the 3′ end of the choC ORF) primers.The intron sequence was verified by comparing the sequence of cDNA with the DNA sequence of the choC ORF.

Cell dry weight and alamar blue assays
For the quantification of mycelial dry weight, the mycelia were harvested from the liquid medium during cultivation every alternate day.Samples were filtered through nitrocellulose filters (pore size 0.45 µm), washed with distilled water, and freeze-dried until constant weight was measured.Cell viability was measured as a percentage of reduction in alamar bluewith absorbance values determined at 570 and 600 nm.Detailed procedures and calculation methods have been described previously (26).

Evans blue staining
Mycelia collected from the MM liquid medium at a certain time (37°C, 120 rpm) were treated with 0.1% Evans blue for 5 min at room temperature, washed three times with phosphate buffered saline (PBS), and then placed on slides for examination under bright-field illumination with an Eclipse 80i microscope (Nikon).

Nucleic acid manipulation
For genomic DNA preparation, mycelia collected were ground into powder using liquid nitrogen, then treated with 500 µL of breaking buffer (8% sucrose, 5% Triton X-100, 50 mM EDTA, 50 mM Tris-Cl, pH 8.0) per 20 mg and incubated at 65°C for 30 min.This was followed by the addition of 500 µL of phenol:chloroform:isoamyl alcohol (25:24:1) to purify the genome.Isopropyl alcohol was added to precipitate the DNA at −20°C.The precipitate collected after centrifugation was resolved in 150 µL distilled water and treated with RNaseA.Genomic DNA was obtained following a second purification step.
To isolate total RNA, 1 mL of TRIZOL reagent was added to each tube with 20 mg of mycelia powder for 10 min, followed by 200 µL of chloroform to purify the nucleic acids.Nucleic acids were precipitated by adding isopropyl alcohol at −20°C and treated with DnaseI (5 U/µL) to get rid of the genomic DNA.The mixture was treated once with phenol:chloroform:isoamyl alcohol (25:24:1) and then once with chloroform:isoamyl alcohol (24:1) until proteins, polysaccharides, and other impurities were eliminated.Finally, RNA was isolated by adding one-tenth volume of 3 M NaAc and twofold volume of absolute ethanol in a −80°C freezer for 20 min and resolved with RNase-free water for reverse transcription reactions.Northern blot analysis was performed as described (63).For northern blotting, choC probes were designed using the genomic DNA of A. fumigatus 293.1 as a template.The probes were labeled with 32 P-dCTP and used for hybridization in a modified Church buffer as previously described (64).About 10 µg of total RNA was separated by elec trophoresis using a 1% agarose gel containing 6% probes.Primer pairs used in this study are listed in Table S4.

Clustering and sequencing
Clustering of the index-coded samples was performed on a cBot Cluster Genera tion System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina, USA) according to the manufacturer's instructions.After cluster generation, library preparations were sequenced on an Illumina HiSeq 2500 platform, and 125/150 bp paired-end reads were generated.

Construction of ΔchoC and choC complementation strains
DJ-PCR was used to construct the ΔchoC mutant.The 5′-flanking and 3′-flanking regions of choC gene were amplified from the genomic DNA of A. fumigstus 293.1 with primer pairs oligo 3,995-3997 and oligo 3,996-3,998.A. nidulans pyrG gene was amplified from A. nidulans FGSC4 using the primer pair oligo 3,868-3,869.The third-round product of DJ-PCR was amplified using the primer pair oligo 3,999-4,000.The A. fumigatus DJ-PCR fragments were purified and used to transform protoplasts generated using the Vino Taste Pro lysis enzyme (65).A solid medium without uracil or uridine was used to select the pyrG + transformants.PCR and restriction enzyme digestion were used to verify the correct transformants.Three A. fumigatus choC deletion mutants were obtained.The choC gene was amplified from the genome of A. fumigatus 293.1 using the primer pairs 4,434-4,435.The A. fumigatus choC gene was cloned into pPTR1 with SmaI digestion.Protoplasts of a ΔchoC strain were transformed with the recombinant pPTRI-choC.Solid MM with 1 µg/mL pyrithiamine was used to select the transformants.We used diagnostic PCR reactions to confirm the correct recombination of the plasmid.Finally, through functional phenotypic validation, three A. fumigatus ΔchoC::choC complemented strains were obtained.

Knockout of CK1, CK2 in WT and ΔchoC strains
The deletion vector pOSCAR-Gcn2 was constructed using the OSCAR protocol described previously (66).Approximately 1-kb fragments from upstream and downstream of the target genes CK1/CK2 were amplified using PCR with the primer sets listed in Table S4, and A. fumigatus 293.1 genomic DNA as the template.The amplified fragments were assembled by introducing PCR products for each gene into the pA-Hyg-OSCAR marker vector and pOSCAR using the BP Clonase II enzyme (Invitrogen, Carlsbad, CA, USA).Then, the reaction mixture was transformed into E. coli DH5α.Bacterial colonies were obtained on LB plates with 100 µg/mL spectinomycin, following overnight incubation at 37°C.Disruption of CK1/CK2 in A. fumigatus was achieved via A. tumefaciens-mediated genetic transformation.pOSCAR-CK1/pOSCAR-CK2 were transformed into A. tumefaciens LBA4404 using the protocol described previously (67).Then, the right transformants were cocultured with 10 7 A. fumigatus 293.1 conidia at 37°C on a nitrocellulose filter that was spread on an IM plate supplemented with 100 mg/L hygromycin and 40 mg/L acetosyringone (Sigma, St. Louis, MO, USA).After induction for 2 days, the filter was transferred onto an MMY plate supplemented with cefotaxime (100 mg/L) followed by incubation for 2 days at 37°C for sporulation.Individual fungal transformants were obtained by single-spore isolation.Two pairs of primers, CK1-5′ Sense/Hygas and Hygs/ CK1-3′ Anti-sense, were used to verify the construct of ΔchoC strain.

RNA-seq analysis
Clean data (clean reads) were obtained by removing reads containing adapters, poly-N, and low-quality reads from the raw data (raw reads) using in-house Perl scripts.Simultaneously, the Q20, Q30, and GC contents of the clean data were calculated.All downstream analyses were based on clean, high-quality data.The reference genome and gene model annotation files for A. fumigatus were downloaded directly from the website (http://ensemblgenomes.org/).A reference genome index was built using Bowtie v2.2.3, and clean paired-end reads were aligned to the reference genome using TopHat v2.0.12.HTSeq v0.6.1 was used to count the number of reads mapped to each gene.The fragments per kilobase per million base pairs sequenced of each gene were then calculated based on the length of the gene and the read count mapped to the gene.Differential expression analysis of three biological replicates per sample was performed using the DESeq R package (version 1.18.0).The resulting P-values were adjusted using Benjamini and Hochberg's approach to control the false discovery rate.Genes with an adjusted P-value < 0.05, found by DESeq, were considered differentially expressed.GO enrichment analysis of differentially expressed genes was performed using the GO seq R package, in which the gene length bias was corrected.GO terms with corrected P-values < 0.05 were considered significantly enriched by differentially expressed genes.We used the KOBAS software to test the statistical enrichment of differentially expressed genes in the KEGG pathways.The protein-protein interaction (PPI) analysis of differentially expressed genes was based on the STRING database, which contains known and predicted protein-protein interactions.For species existing in the database, we constructed networks by extracting the target gene list from the database; otherwise, Blastx (v2.2.28) was used to align the target gene sequences to the selected reference protein sequences, and then the networks were built according to the known interactions of the selected reference species.

Lipid extraction
A certain amount of hyphae of each sample (30 mg) was mixed with acetonitrile, and the mixture was kept at 4°C for 4 h.A quantity of 200 µL of extract was taken, and 500 µL of CHCl 3 -CH 3 OH (2:1, vol/vol) was added, vortexed, and 200 µL of water was subsequently added.The mixture was centrifuged to remove the organic layer, dried, and resolved with 200 µL of acetonitrile-isopropanol for testing.

High performance liquid chromatography-mass spectrometry (HPLC-MS)
Chromatography was performed on an ACQUITY UPLC-Xevo G2 Q-TOF UPLC system (Waters, USA).Briefly, the compounds were eluted from a UPLC BEHC18 column (Waters, 1.7 µm, 2.1 × 100 mm) in an isocratic gradient consisting of 60% (A) ultrapure water/ acetonitrile (4:6, vol/vol) and 40% (B) acetonitrile/isopropanol (1:9, vol/vol) containing 0.1% formic acid and 5 mM acetamide as mobile phases.The flow rate and column temperature were set at 0.3 mL/min and 55°C, respectively.The LC system was coupled to a Q-TOF tandem mass spectrometer fitted with an electrospray ionization (ESI) source.The ESI ion source has a mass scanning range of 50-1,200 m/z.Nitrogen was used for all gas paths.The operating parameters are listed in Table 2.The compounds were monitored in both positive and negative ion modes.The software package Masslynx 4.1 was used for mass spectral data acquisition and quantitation.

Data processing
Conversion of mass spectrometry data was carried out using MZmine software after the following steps: denoising, mass spectral peak extraction, deconvolution processing, peak arrangement, alignment, merging, listing in Excel and re-denoising, gap filling, data export, and other processes.The final data format included ID, m/z, RT, and (sample number) integral area (the original data can be found in Tables S1 to S8).Data analysis could be performed using raw or normalized processed data.

Survival and fungal burden tests
BALB/c mice were given free access to water and food and were maintained in a temperature-controlled (22°C) room with a 12/12 h light/dark cycle (8:00 a.m., lights on; 8:00 p.m., lights off).Mice received an intravenous injection of 0.1 mL of the inocula and were monitored for 3 weeks after the injection.The mortality rate was recorded daily.Liver, spleen, and kidney tissues from mice infected with different strains were removed and homogenized with an appropriate volume of 1× PBS.Primary homogenate dilutions were quantitatively cultured by serial dilution, plated on MMY plates, and incubated at 37°C for 24-36 h, after which A. fumigatus fungal burdens (numbers of CFU per gram of lung tissue) were determined.

Histopathological study
Infected animals were sacrificed on days 3, 13, and 23 after inoculation.The lungs, liver, spleen, and kidneys were fixed in formalin, and paraffin-embedded sections were stained with HE and PAS and photographed under a light microscope.

Data analysis
All experiments were randomized, and the scientists who performed the quantification were blinded.Statistical analyses were performed using analysis of variance (ANOVA) and Student's t-test, unless specified otherwise, using GraphPad software.Student's t-tests were used to compare the two conditions.A one-way ANOVA was used to compare multiple experimental conditions.The Bonferroni post hoc test was used to compare each condition.For dry weight and alamar blue reduction analysis, a two-way ANOVA was used for comparisons at different time points.All data are shown as the mean ± SEM.Statistical significance was set at P < 0.05 significant.

FIG 2
FIG 2 Roles of choC in cell viability, cell death, and cell wall integrity.(A) Mycelial dry weights of WT and ΔchoC strains grown in liquid MM, MM + DAE, and MM + choline at 37°C for 9 days.Data are the mean values of three independent experiments.(B) Phenotypes of mycelial aggregates formed in liquid MM at days 2 and 6.Scale bar: 20 µm (middle panels) and 10 µm (bottom panels).(C) Alamar blue reduction rates of WT and ΔchoC strains grown in liquid MM, MM + DAE, and MM + choline at 37°C for 9 days.Data points are the mean of three independent experiments.(D) Apoptosis analyses of WT and ΔchoC strains grown in liquid MM by Evans blue staining.Scale bar is 5 µm.The photographs were taken on days 2 and 6. (E) Sensitivity to osmotic pressure of WT and ΔchoC strains grown in liquid MM with 1.2 M sorbitol.Scale bar is 10 µm.(F) Effects of Congo Red on the growth of the ΔchoC mutant.Scale bar: 10 and 20 µm.

FIG 3
FIG 3 DEGs and gene ontology (GO) enrichment analyses.(A) Heatmap analysis shows that statistical significance in some important genes in phospholipid metabolism, cell cycle, autophagy, and other metabolic pathways in WT-2,ΔchoC-2 and WT-4,ΔchoC-4 strains.WT-2/ΔchoC-2 means WT/ΔchoC strains were cultured in MM for 2 days.WT-4/ΔchoC-4 means WT/ΔchoC strains were cultured in MM for 4 days.Triplicates were analyzed per strain per time point.(B) GO enrichment analysis of DEGs between ΔchoC-2 and WT-2.(C) GO enrichment analysis of DEGs between ΔchoC-4 and WT-4.(D) GO enrichment analysis of DEGs between ΔchoC-4 and ΔchoC-2.(E) GO enrichment analysis of DEGs between WT-4 and WT-2.The ordinate is an enriched GO term, and the abscissa is the number of DEGs in the term.Different colors are used to distinguish three major categories: biological processes, cell components, and molecular functions."*" is a significant enrichment of GO term.

FIG 4
FIG 4 Statistical analysis of KEGG pathway enrichment.(A) The metabolic pathways of significantly upregulated DEGs in the ΔchoC mutant at 2 days.(B) The metabolic pathways of significantly downregulated DEGs in the ΔchoC mutant at 2 days.(C) The metabolic pathways of significantly upregulated DEGs in the ΔchoC mutant at 4 days.(D) The metabolic pathways of significantly downregulated DEGs in the ΔchoC mutant at 4 days.Rich factor refers to the ratio of the

FIG 5
FIG 5 Influence of choC on lipid metabolism in A. fumigatus.(A) The abundance of PC in WT and ΔchoC strains at days 2 and 4. (B) The abundance of PE in WT and ΔchoC strains at day 2. N = 6, *P < 0.05; **P < 0.01; and ***P < 0.001.(C) The abundance of PE in WT and ΔchoC strains at day 4. N = 6, *P < 0.05; **P < 0.01; and ***P < 0.001.(D) The abundance of TAG in WT and ΔchoC strains at day 4. (E) The abundance of DAG in WT and ΔchoC strains at days 2 and 4.

FIG 6
FIG 6 Requirement of ChoC for the pathogenesis of A. fumigatus.(A) Survival curve of mice infected with A. fumigatus in 3 weeks.(B) Fungal burden of the liver in mice infected with A. fumigatus at days 3, 10, and 17. (C) Fungal burden of the spleen in mice infected with A. fumigatus at days 3, 10, and 17. (D) Fungal burden of the kidney in mice infected with A. fumigatus at days 3, 10, and 17. (E) Representative images of periodic acid-Schiff (PAS) and hematoxylin-eosin (HE)-stained kidney sections (200×) on day 23.Bar = 100 µm for 200.

TABLE 1
Strains used in this study

TABLE 2
Operating parameters of mass spectrometry