CsdA-LaeB hub governs Aspergillus fumigatus virulence via FqC biosynthesis

CsdA-LaeB hub governs Aspergillus fumigatus virulence via FqC biosynthesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article CsdA-LaeB hub governs Aspergillus fumigatus virulence via FqC biosynthesis Wenbing Yin, Zili Song, Hongjiao Zhang, Leixin Ye, Yuxin Lei, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6615529/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The intricate interplay between secondary metabolism and fungal pathogenesis remains incompletely understood. Here, we uncover a regulatory hub in Aspergillus fumigatus that coordinates virulence through a specialized metabolite network. Using a multi-omics combined chemistry strategy, we identified fumiquinazoline C (FqC) as a keystone metabolite enabling fungal pathogenicity. Central to this process is the RNA-binding protein CsdA, which forms a dynamic nuclear complex with the global regulator LaeB, bridging metabolic remodeling to virulence. Disrupting this complex led to hyperactivation of secondary metabolism, enhancing fungal colonization and virulence. Through deconstructing this hub of CsdA-LaeB-FqC, we pinpointed PptA—a phosphopantetheinyl transferase essential for secondary metabolite synthesis—as a linchpin controlling metabolic virulence. Strikingly, FDA-approved drugs (tepotinib, ibrutinib, eltrombopag) targeting PptA suppressed FqC biosynthesis, reduced fungal burden and attenuated lung inflammation in murine models significantly. These findings decode a pathogen’s “metabolic virulence code” and establish a drug-repurposing paradigm to combat antifungal resistance Biological sciences/Microbiology/Fungi Biological sciences/Chemical biology Figures Figure 1 Figure 2 Figure 3 Figure 5 Figure 6 Main Invasive aspergillosis, a life-threatening fungal infection primarily caused by the opportunistic pathogen Aspergillus fumigatus , represents a significant global health burden and remains a leading cause of mortality 1 – 5 . Annually, over 2 million individuals develop invasive aspergillosis, primarily in the context of chronic obstructive pulmonary disease (COPD), intensive care, lung cancer, or hematological malignancies, resulting in an estimated crude annual mortality of 1.8 million 6 . A. fumigatus , a formidable human pathogen, predominantly infects immunocompromised individuals, such as cancer patients, transplant recipients, and those grappling with COPD 7 – 10 . To successfully invade the host, A. fumigatus employs a range of virulence strategies, including thermotolerance, cell wall modifications, nutritional adaptability, stress responses, and interactions with the host immune system 9 , 11 – 14 . Notably, during host-pathogen interactions, bioactive secondary metabolites (SMs) produced by the invading A. fumigatus play a crucial role in modulating immune responses 15 , 16 . Examples include DHN-melanin, which confers resistance to reactive oxygen species (ROS), and gliotoxin, which impairs immune cell function 17 – 19 . However, the regulatory networks governing their biosynthesis during infection, as well as their potential as therapeutic targets, remain poorly understood. Fungal SMs are encoded by biosynthetic gene clusters (BGCs). In A. fumigatus , 20 BGCs have been linked to specific SMs 20 . SM production is regulated by complex hierarchical networks, that include pathway-specific, epigenetic, and global regulation 19 , 21 , 22 . Several pathway-specific transcription factors (e.g., GliZ for gliotoxin), epigenetic modifiers (e.g., histone deacetylase RpdA) and global regulators (e.g., LaeA, control approximately 50% BGCs) have been characterized 23 – 26 . RNA-binding proteins (RBPs) emerge as a novel class of regulators influencing eukaryotic gene expression, are well studied in human disease 27 , 28 and plant development 29 , 30 , yet their roles in fungal metabolism and virulence are veiled in an enigma. Furthermore, although global regulators such as LaeB are known to coordinate BGC activation 31 , 32 , their interplay with RBPs and relevance to clinical isolates remain uncharted. Here, we bridge this gap by integrating pan-metabolomics, functional genetics, and drug repurposing to unravel a novel regulatory hub driving A. fumigatus virulence. Through pan-metabolome analysis, we identified distinct metabolic profiles between clinical and environmental A. fumigatus isolates, with fumiquinazoline C (FqC) emerging as a major contributor to virulence. Transcriptomic and metabolomic analyses revealed an unknown interaction between CsdA and LaeB, confirmed by in vitro pull-down and in vivo bimolecular fluorescence complementation assays. Strikingly, disruption of this complex hyperactivated FqC production, increasing fungal colonization and virulence, revealing a delicate balance between metabolic regulation and host adaptation. Importantly, we identified Sfp-type PPTases — a conserved class of phosphopantetheinyl transferases critical for widespread SMs biosynthesis — as a universal target for anti-infective therapy. We further demonstrated that tepotinib, ibrutinib, and eltrombopag inhibit FqC synthesis, and consequently reducing fungal colonization and inflammation in mouse lungs. This study provides an insightful perspective on the metabolic regulation of fungal pathogenesis and proposes a novel anti-infective strategy targeting global secondary metabolism. Results Metabolic divergence between clinical and environmental A. fumigatus isolates To explore the link between A. fumigatus metabolism and virulence, we performed a non-targeted pan-metabolomic analysis of 19 A. fumigatus isolates, including 5 environmental and 13 clinical strains, as well as model strain CEA17 (as also clinical isolate) (Extended Data Fig. 1 and Supplementary Table 1). Total ion peaks give a good indication of the relative number of metabolites produced. Principal components analysis (PCA) revealed significant metabolic differences between clinical and environmental isolates (Fig. 1 a). To quantify the metabolic differences between the two groups, we compared the abundance of each detected ion across isolates using the environmental isolate 34 as a reference. Quantitative profiling revealed 11%-12% of detected metabolic ion products exhibited abundance variations in all other environmental isolates. Strikingly, clinical isolates demonstrated substantially greater divergence, with 25%-64% of metabolic ion products differing from environmental isolate 34, including a 61% variation in the model strain CEA17 (Fig. 1 b). These findings suggest a link between global metabolism and A. fumigatus virulence. CsdA governs global metabolism and virulence Fungal metabolism is usually controlled by a series of key regulatory proteins. We recently discovered an RNA-binding protein (RBP) CsdA in the endophytic fungus Pestalotiopsis fici that is critical for normal growth and metabolism 33 . RBPs play crucial roles in regulating pathogenicity in various eukaryotes, yet their mechanisms of action in fungal pathogenicity remain unclear. Analysis of fungal genome data revealed CsdA homologues existing in fungal kingdom especially in pathogenic fungi (Supplementary Fig. 1), including Aspergillus spp. , Histoplasma capsulatum , Blastomyces gilchristii , and dermatophytes ( Arthroderma uncinatum, Trichophyton rubrum, T. mentagrophytes ), Candida auris and C. albicans . Interestingly, the identity/coverage of CsdA homology in common fungal pathogen B. gilchristii was 83.76%/100% relative to the A. fumigatus homolog. Basidiomycete fungi ( Cryptococcus neoformans var. grubii ) exhibited lower identity/coverage (30%/10%) but retained similar RNA recognition motif (RRM) and zinc finger (ZnF) domains (Supplementary Fig. 1 and Supplementary Table 2). This suggests that CsdA is prevalent in Ascomycota, and notably in fungal pathogens. To examine the function of CsdA, a comparative metabolome analysis between the Δ csdA mutant and control CEA17 strain revealed that 1,321 (26% of total) ion peaks were significantly regulated among the detected 5,057 products (adjusted p 1) (Fig. 1 c and Supplementary Table 3). To mimic the host’s lung environment, we compared the growth and metabolite profiles of Δ csdA mutant between RPMI1640 medium (a commonly used cell medium) and lung plate (agar supplemented mouse lung homogenate) (Fig. 1 d). This mutant showed a faster growth in the lung plate compared to the RPMI1640 medium (Fig. 1 e). Through a comparative metabolomics analysis, we found that the metabolic ion products regulated by CsdA in RPMI1640 medium and lung plate were significantly higher than GMM medium (Fig. 1 c, f). Among them, 3,252 (75% of total) ion peaks in RPMI1640 medium were regulated by CsdA, including 1,510 (35%) down-regulated and 1,742 (40%) up-regulated (Fig. 1 f). Similarly, 3,042 (78% of total) ion peaks in lung plate were regulated by CsdA, including 1,593 (41%) down-regulated and 1,449 (37%) up-regulated (Fig. 1 f). Given the established role of A. fumigatus toxins in virulence 15 , we presumed that metabolites regulated by CsdA might impact the virulence of this pathogen. In a neutropenic murine model of invasive aspergillosis (Fig. 1 g), infection with the Δ csdA mutant resulted in 100% mortality by day 8, compared to 70% in the control strain. Complementation of csdA restored mortality to wild-type levels (Fig. 1 h). Meanwhile, the Δ csdA mutant also had a higher fungal burden in infected murine lungs, concomitant with the higher mortality (Fig. 1 i, j). Histological examination revealed extensive lung tissue damage in Δ csdA -infected mice, characterized by the extensive conidial germination and invasive mycelium formation. In contrast, the control strain only exhibited limited pulmonary epithelial invasion (Fig. 1 k, l). CsdA interacts with LaeB to orchestrate metabolism and virulence Previously, we demonstrated that CsdA interacts with RsdA to regulate secondary metabolism in P. fici 33 . Upon systematic investigation, RsdA’s ortholog protein with 49%/80% identity/coverage was found in A. fumigatus . Interestingly, the protein is also identified in other Aspergillus species including A. flavus 32 and A. nidulans 31 , known as LaeB (Fig. 2 a and Supplementary Fig. 2). In these species, LaeB, a protein with undefined domains, has an established role in regulating secondary metabolism, although its function in A. fumigatus remains uninvestigated. Thus, a Δ laeB mutant was constructed in the CEA17 background to characterize its function (Fig. 2 b). Similar to the Δ csdA mutant, a comparative metabolomics analysis revealed a total of 5,045 ion peaks were detected, among which 2,083 (41%) displayed significant changes in the Δ laeB mutant (adjusted p 1) (Fig. 2 c and Supplementary Table 3). Notably, 1,575 (31%) were significantly down-regulated, while 508 (10%) were significantly up-regulated (Fig. 2 c). In the neutropenic murine model, we observed that the Δ laeB mutant – similar to the Δ csdA mutant – also displayed a 100% fatality rate at 7 days after infection, and the laeB complement strain was restored to near WT levels of virulence (Fig. 2 d). Histological analysis of the murine lungs infected with the Δ laeB mutant also exhibited severe pathology characterized by invasive mycelium, along with a significantly heightened fungal burden when compared to the control strain (Fig. 2 e, f). This suggests a fascinating connection of LaeB and CsdA in mediating fungal virulence. To probe the relationship between CsdA and LaeB, we analysed the subcellular localization of CsdA and LaeB in A. fumigatus (Fig. 2 b). Fluorescent labelling of both proteins (LaeB-sfGFP and CsdA-mCherry) coupled with DAPI (4’,6-diamidino-2-phenylindole, a DNA binding dye) staining revealed that CsdA and LaeB co-localize in the nucleus (Fig. 2 g and Extended Data Fig. 2 a). Next, we expressed LaeB-His and GST-CsdA fusions in bacteria and used them in pull-down assay. GST-CsdA (92.4 kDa) was co-purified when LaeB-His (89.6 kDa) was pulled down using anti-His antibody, suggesting a direct physical interaction between LaeB and CsdA (Extended Data Fig. 2 b-d). To confirm the interaction between the two proteins in the nucleus, we created laeB-YFP N and csdA-YFP C fusions in A. fumigatus and used them in a bimolecular fluorescence complementation (BiFC) assay. We observed a yellow fluorescent signal indicating that the two proteins interact physically and the signal co-localized with DAPI indicating that the interaction happens in the nucleus (Fig. 2 h). Together, these results demonstrate that CsdA and LaeB interact to form a functional complex. CsdA/LaeB regulates key secondary metabolites, including FqC To investigate how loss of either CsdA or LaeB increases virulence, we constructed a regulatory network co-mediated by both proteins, involving co-regulated metabolic ions and genes (Fig. 3 a). Venn analysis identified 809 metabolic ions with significantly altered abundances in both Δ csdA and Δ laeB mutants (Fig. 3 b). Transcriptomic analysis revealed that 2,380 (21%) and 2,789 (24%) genes exhibited over twofold higher expression in the Δ csdA and Δ laeB mutants, respectively, compared to the control strain (Fig. 3 b and Supplementary Table 4). A total of 911 genes were significantly regulated in both mutants, predominantly related to secondary metabolism (Fig. 3 c). Since most secondary metabolites (SMs) are encoded by biosynthetic gene clusters (BGCs), we mapped gene expression profiles of all 39 BGCs in the A. fumigatus genome 20 , 34 (Fig. 3 d and Extended Data Fig. 3 a). Of these, 15 (39%) backbone genes were significantly co-regulated in both mutants, with 13 displaying the same expression trend, including 3 up-regulated and 10 down-regulated, such as fmqC , encA , gliP , afumA , nscA , etc . (Extended Data Fig. 3 b-d). In addition to gliotoxin 35 – 37 , A. fumigatus produces multiple mycotoxins 15 that modulate host immunity, such as endocrocin 38 and fumiquinazoline C (FqC) 39 . To explore how CsdA and LaeB regulate these specific mycotoxins, we systematically analysed the BGCs controlled by the two proteins and categorized them based on the expression intensity of their backbone genes. Among the most prominently affected BGCs upon csdA deletion, gliotoxin, fumiquinazoline, and endocrocin biosynthesis genes were up-regulated, while fumihopaside and neosartoricin biosynthesis genes were significantly down-regulated (Fig. 3 e, f). Specifically, fumiquinazoline production involves five genes ( fmqA-E ), with four of them ( fmqA-D ) negatively regulated by the trans -acting transcription factor SebA 39 . In both Δ csdA and Δ laeB mutants, fmqB - D were significantly up-regulated, while sebA was significantly down-regulated. Metabolomics analysis revealed a dramatic 19.84- and 13.35-fold increase of FqC production in the Δ csdA or Δ laeB mutants, respectively, compared to the control strain (Fig. 3 g). This suggested that CsdA and LaeB co-regulate the expression of fmqB-D through SebA, and ultimately influencing FqC production. In contrast, the immunosuppressant gliotoxin production was only subtly up-regulated by 2.11- and 2.77-fold in the Δ csdA and Δ laeB mutants, respectively, compared to the control (Fig. 3 h). The biosynthesis of gliotoxin involves 13 genes within the cluster and one transcription factor RglT outside the cluster 23 , 40 . In both mutants, the expression of gliM and gliK , along with 12 other genes (includin gliZ and rglT ), was up-regulated to varying degrees (Fig. 3 h). Based on current understanding of the endocrocin biosynthetic pathway 41 , four genes ( encA-D ) in the cluster are involved. Transcriptome analysis revealed the upregulation of four genes by more than twofold in both mutants, with LaeB exerting more significant regulation than CsdA (Extended Data Fig. 3 e). Conversely, all gene expressions in the neosartoricin and fumihopaside BGCs were significantly down-regulated in both mutants (Extended Data Fig. 3 f, g). Collectively, although CsdA/LaeB orchestrates multiple mycotoxins, FqC emerges as the predominant regulated metabolite. FqC is a key virulence metabolite To further gain additional support for the regulation of secondary metabolism contributing to virulence in the csdA and laeB deletion mutants (Fig. 4 a), we measured the expression levels of csdA and laeB as well as their co-regulated BGC backbone genes by qRT-PCR across clinical strains. The results showed that csdA and laeB expression were significantly down-regulated in clinical strains compared to the control strain CEA17, with laeB exhibiting greater repression (Fig. 4 b, c). Interestingly, the backbone gene expression and production of FqC were significantly up-regulated across multiple clinical strains compared to the control ( p < 0.05), suggesting a potential role for this metabolite in A. fumigatus pathogenesis (Fig. 4 d, e). We simulated the culture of clinical strains in mouse lung homogenates to identify the key pathogenic metabolites. Compared with control CEA17, FqC production in other clinical strains were up-regulated by more than twofold (Fig. 4 f and Extended Data Fig. 4 ). These results further support the role of FqC in A. fumigatus pathogenicity. Subsequently, a Δ fmqC mutant was constructed in the same CEA17 background (Fig. 4 g and Extended Data Fig. 5 ), and its virulence compared to the Δ csdA mutant and wild-type strains. The Δ fmqC mutant exhibited significantly attenuated virulence, with 60% mortality at 8 days’ post-infection compared to 90% in wild-type strain ( p < 0.05, Fig. 4 h). Quantitative fungal burden analysis revealed reduced lung colonization in mice infected with Δ fmqC mutant compared with wild-type strains ( p < 0.001, Fig. 4 i). These findings suggest that CsdA-mediated pathogenesis may be primarily attributable to its regulation on FqC biosynthesis. To explore whether FqC serves as the key metabolite underlying CsdA- and LaeB-induced virulence, we generated Δ csdA Δ fmqC and Δ laeB Δ fmqC double mutants in the CEA17 background (Extended Data Fig. 5 ) and compared their virulence to the Δ fmqC single mutant and wild-type strains. Metabolic profiling revealed that neither the Δ csdA Δ fmqC and Δ laeB Δ fmqC double mutants nor the Δ fmqC single mutant produced FqC, compared to the control strain (Fig. 4 g). Critically, the mortality of Δ csdA Δ fmqC and Δ laeB Δ fmqC double mutants was restored to wild-type levels, compared to that of the single mutants (Fig. 4 j). Furthermore, there were no significant differences in fungal colonization or histopathology between the double mutants and the control strains ( p > 0.05, Fig. 4 k, l). These findings establish FqC as the key effector of CsdA/LaeB-mediated pathogenicity. Targeting Sfp-type PPTase for antifungal therapy Multiple SMs contributes to the overall A. fumigatus pathogenicity 15 . To dismantle A. fumigatus pathogenicity at its metabolic core, we focused on Sfp-type PPTase—a 4’-phosphopantetheinyl transferase essential for synthesizing polyketides (PKs) and non-ribosomal peptides (NRPs), two major virulence-associated SM classes, known as PptA in A. fumigatus 42 – 44 (Fig. 5 a and Extended Data Fig. 6 a). Strikingly, deletion of pptA in the CEA17 background revealed near-complete disruption of hyphal growth, asexual development, and global secondary metabolism, demonstrating its potential as a potent antifungal target (Extended Data Fig. 6 b-d). Subsequently, we predicted PptA structure using Alphafold2, and performed virtual screening of 3,019 marketed drugs in the FDA-Approved Drug Library (Fig. 5 a and Supplementary Fig. 3). According to the affinity between PptA and ligands, all candidate drugs were scored and ranked, and the antifungal activities of the top eight drugs were evaluated in vitro (Fig. 5 a, Supplementary Figs. 4,5 and Supplementary Table 5). Three drugs—tepotinib, ibrutinib, and eltrombopag—demonstrated significant growth inhibition against A. fumigatus , with inhibition ratios of 67%, 27%, and 36%, respectively (Fig. 5 b-d). To validate PptA targeting, we performed qRT-PCR assay, revealing 2.8-, 5.2-, and 4.9-fold reductions in pptA expression following three drug treatments ( p < 0.0001), respectively (Fig. 5 e). Meanwhile, we also analysed the metabolic ionic products of the drug-treated CEA17 strain (Supplementary Table 6). Crucially, they broadly compromised SM production, reducing FqC levels by 1.3–2.2-fold ( p < 0.01) and globally suppressing 32–53% of metabolic ion products (Fig. 5 f, g, Extended Data Figs. 7–9 and Supplementary Fig. 6). The therapeutic potential of this strategy was validated across 13 clinical A. fumigatus isolates, with all three tested drugs demonstrating varying degrees of growth and metabolic inhibition (Fig. 5 h-j). Notably, tepotinib exhibited the most potent activity, achieving up to 70% growth inhibition and reducing FqC production by up to 6.3-fold ( p < 0.0001). In murine models of invasive aspergillosis, intranasal discontinuous administration for 5 days reduced pulmonary fungal burden by 1.7 to 2.1-fold ( p < 0.05) and attenuated tissue damage (Fig. 5 k, l), demonstrating in vivo efficacy against A. fumigatus infection. These findings reveal targeting secondary metabolic biosynthetic pathway as a promising therapeutic strategy for combating invasive aspergillosis. Broad-spectrum antifungal activity of drug candidates Given the ubiquitous presence of SMs across fungi, we conducted a comprehensive phylogenetic analysis of PptA orthologs (Supplementary Fig. 7). Evolutionary conservation analysis revealed that PptA orthologs are widely distributed among fungi, with four critical drug-binding sites (D159, W227, E231, and K235) showing remarkable conservation (Fig. 6 a, b, Extended Data Fig. 6 e and Supplementary Fig. 8), suggesting potential broad-spectrum antifungal activity of drug candidates. Then, we evaluated the efficacy of tepotinib, ibrutinib, and eltrombopag against divergent fungi ( A. flavus , Mucor circinelloides , Fusarium oxysporum , C. neoformans, C. gattii ). Tepotinib emerged as the most potent agent against A. flavus , inhibiting growth by 57% and metabolites production by 52% ( p < 0.05, Fig. 6 c and Supplementary Fig. 9). Ibrutinib showed maximal efficacy against F. oxysporum and M. circinelloides , achieving 73% and 68% growth suppression, as well as 37% and 42% metabolic inhibition ( p < 0.01, Fig. 6 d, e, Supplementary Fig. 9), respectively. Strikingly, eltrombopag demonstrated maximal efficacy against Cryptococcus (Fig. 6 f), and significantly inhibiting metabolism in both C. neoformans H99 (64% reduction) and C. gattii R265 (59% reduction) ( p < 0.0001, Fig. 6 g, h). To enhance clinical relevance, we combined these agents with amphotericin B (AmB), a cell membrane-targeting fungicide 45 . Compared to standalone IC 50 values of AmB (0.92 µM), tepotinib, ibrutinib, and eltrombopag showed higher concentration (100.4–765.9 µM) against A. fumigatus . Strikingly, combination with AmB significantly enhanced efficacy (0.12–0.35 µM), with combination index of 0.14–0.38 (Fig. 6 i, j and Extended Data Fig. 10). This dual-target strategy—simultaneously disrupting metabolic virulence and cell integrity—represents a paradigm shift in circumventing antifungal resistance. Discussion Although A. fumigatus pathogenicity has been characterized at genomic and transcriptomic levels 3 , 9 , 46 , the contribution of global secondary metabolism to infection remains enigmatic 22 , 47 . Fungal pathogenicity has traditionally been attributed to thermotolerance 9 , 11 , 48 , cell wall modifications 12 , 49 , nutritional adaptability 50 – 52 , biofilm 53 , 54 , and stress responses 55 , 56 . However, our findings reveal an additional layer of pathogenesis: the remodeling of secondary metabolism to coordinate virulence. Here, we identify the CsdA-LaeB complex as a master conductor of this process, bridging SM dynamics to virulence. Integrative metabolomic profiling of clinical versus environmental isolates uncovered FqC as a clinically enriched SM that is critical for lung invasion and establishment of invasive aspergillosis. Since the biosynthetic genes of fumiquinazoline were identified in 2010 57 , its role in virulence remained unknown until this study. Pan-metabolomics analysis of clinical and environmental isolates of A. fumigatus demonstrated differences in metabolic ion abundance between the two groups (Fig. 1 a, b). Notably, FqC was significantly enriched in clinical strains and negatively regulated by CsdA, suggesting its involvement in the CsdA-mediated regulatory pathway controlling of A. fumigatus virulence (Figs. 3 g, 4 d-f). This was subsequently demonstrated by constructing double mutants of Δ fmqC Δ csdA and Δ fmqC Δ laeB and in a murine model of invasive aspergillosis (Fig. 4 j-l). Genetic dissection revealed that CsdA and its nuclear interactor LaeB that orchestrates FqC biosynthesis and virulence in A. fumigatus by forming a regulatory hub CsdA-LaeB-FqC. Disruption of the complex (Δ csdA or Δ laeB ) triggered a 13.4–19.8-fold FqC surge (Fig. 3 g), correlating with a rise in murine mortality (up to 100% by day 7 post-infection, Figs. 1 h, 2 d)—a phenomenon implies evolutionary trade-offs between metabolic dynamics and virulence. Fungal RBPs frequently function within protein complexes to regulate cellular processes 58 . Distinct from the functions of regulating growth and development reported in F. graminearum (FgRbp1-U2AF23) 59 and M. oryzae (RBP35-CFI25) 60 , this discovery positions RBP complex as central players in fungal metabolism and virulence. Metabolomics and transcriptomic analysis showed that 809 metabolic ion products and 911 genes (including 33% BGC backbone genes) were co-regulated by CsdA and LaeB (Fig. 3 b). This is consistent with the regulatory mode of secondary metabolism in the endophytic fungus P. fici 33 . Current efforts to distinguish clinical A. fumigatus strains rely on pan-genomics 61 , 62 or azole-resistance markers 63 , 64 , overlooking metabolic drivers. Our pan-metabolomic profiling of clinical versus environmental isolates identified FqC as a potential diagnostic biomarker and therapeutic target. By targeting PptA—a conserved phosphopantetheinyl transferase essential for widespread SMs biosynthesis (including FqC)—we repurposed three FDA-approved drugs (tepotinib, ibrutinib, eltrombopag) to cripple fungal infection. These drugs inhibited A. fumigatus growth by 27–67% ( p < 0.01), suppressed FqC production by 1.3–2.2-fold ( p < 0.01), and reduced pulmonary fungal burden in invasive aspergillosis mouse models by 1.7–2.1-fold ( p < 0.05, Fig. 5 ). Therapeutic efficacy was validated across 13 clinical A. fumigatus isolates, with all three drugs showing dose-dependent antifungal effects (Fig. 5 h-j). Strikingly, tepotinib demonstrated superior potency, achieving 70% growth inhibition and a 6.3-fold reduction in FqC production ( p < 0.0001). These findings establish PptA as a key controller of SM biosynthesis, including FqC production, and highlight targeting secondary metabolic biosynthetic pathway as a promising therapeutic strategy for combating invasive aspergillosis. While the scientific community is often fixated on the dual - edged nature of SMs, marveling at their roles as either potent natural drugs 22 , 65 or menacing virulence factors 18 , 66 , an entire realm of untapped potential lies hidden: the remarkable promise of their biosynthetic pathways as powerful anti-infection targets. The species-specific biosynthesis of SMs in fungi 22 , underscores their potential as an innovative, highly selective and safe therapeutic target. The high conservation of drug-binding sites (D159, W227, E231, K235) across fungal Sfp-type PPTases highlights its potential as a broad-spectrum antifungal target (Fig. 6 a, b). Among the tested pathogens, tepotinib was the most potent against A. flavus (Fig. 6 c), ibrutinib showed maximal efficacy against F. oxysporum and M. circinelloides (Fig. 6 d, e). Remarkably, eltrombopag exhibited maximal efficacy against Cryptococcus (Fig. 6 f), and significantly inhibiting metabolism in both C. neoformans H99 and C. gattii R265 (Fig. 6 g, h), which may aid in combating fatal fungal infection. Furthermore, three agents also demonstrated potent synergistic effects with amphotericin B, revealing a novel dual-target antifungal strategy that combines conventional cell membrane disruption with secondary metabolism inhibition. These antifungal effects targeting non-traditional targets, combined with dual-target synergy (AmB + PptA inhibitors), establishes a blueprint for combating multidrug-resistant infections. In conclusion, our work redefines antifungal therapy by shifting focus from traditional drug target of cell membrane to metabolic choke points. The CsdA-LaeB-FqC hub illuminates how pathogens dynamically recalibrate virulence metabolites, while Sfp-type PPTase-targeted drug repurposing delivers an actionable strategy to outmaneuver resistance. By deciphering pathogen’s “metabolic virulence code”, we provide both a mechanistic framework and a therapeutic toolkit to combat the escalating threat of invasive fungal diseases. Methods Antibodies Anti-His, Anti-GST and HRP-Goat Anti-Mouse from Proteintech (66005-1-Ig, 66001-2-Ig, SA00001-1) were used for western blot assays. Ethics statement All murine experiments were performed in strict accordance with the “the regulation of the Institute of Microbiology, Chinese Academy of Sciences of Research Ethics Committee.” The murine experiment protocol was approved by the Institute of Microbiology, Chinese Academy of Sciences of Research Ethics Committee (Permit No. APIMCAS2022106). Mice Balb/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Male mice (age, 7–8 weeks) were injected with cyclophosphamide and cortisone acetate to generate a murine model of invasive aspergillosis. Strains and cultivation The strains used in this study are listed in key resources Supplementary Table 7. All Aspergillus fumigatus strains were grown at 37°C on glucose minimum medium (GMM) with appropriate supplements corresponding to the auxotrophic marker or antibiotics 67 , 68 . A. fumigatus and its transformants were cultivated in liquid GMM medium at 25°C for 3 days to extract total RNAs and for 5 days to detect secondary metabolites (SMs). Escherichia coli strains DH5α and BL21 were cultured in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl) supplemented with appropriate antibiotics to construct plasmids and express proteins, respectively. Saccharomyces cerevisiae BJ5464 69 were used for construction of green fluorescent protein (sfGFP) and red fluorescent protein (mCherry) expression vectors on synthetic dextrose complete medium with appropriate supplements corresponding to the auxotrophic markers 70 . Gene cloning and plasmid construction All plasmids and primers (BEIJING TSINGKE BIOTECH Co., Ltd., CHINA) are given in Supplementary Table 7. For the construction of deletion mutants, around 1 kb upstream and downstream fragments of the targeted genes were amplified from A. fumigatus genomic DNA (gDNA) by high-fidelity DNA polymerase TransStart ® FastPfu (TRANSGEN BIOTECH, CHINA). The marker genes AfpyrG and hph were amplified from the vectors pYH-WA-pyrG and pXW55-hph, respectively, and fused with the flanking sequences of the target genes to form different deletion cassettes by the double-joint PCR method described previously 71 . For protein expression, high-fidelity DNA polymerase Q5 (NEW ENGLAND BIOLABS) was used to amplify the open reading frames (ORFs) of targeted genes that were then inserted into pET28a ( His 6 -tag) or pGEX-4T ( GST -tag) to produce pYSZL46 (LaeB-His) or pYSZL47 (GST-CsdA) through the quick-change method 71 . For the subcellular localization of CsdA, the csdA , mCherry , and hph were integrated into the Spe I/ Pml I-cleaved pXW55 vector via the yeast recombination method 72 to give the plasmid pYSZL13 ( csdA-mCherry ). The same method was used to construct sfGFP expression vectors pYSZL11 ( laeB-sfGFP ). In order to construct complementary vectors, csdA and hph were integrated into the Spe I/ Pml I-cleaved pXW55 vector via the Clone Express® MultiS One Step Cloning Kit (VAZYME BIOTECH Co. Ltd, CHINA) to give the plasmid pYSZL56 ( csdA-hph ). The same method was used to construct pYSZL57 ( laeB-hph ). The above plasmids were verified by PCR with 2×GS Taq polymerase (GENESAND, CHINA). To construct bi molecular f luorescence c omplementation (BiFC) vectors, the AfpyrG or hph gene was cloned into the Kpn I/ Hind III-cleaved pCX62-CYFP or pKNT-NYFP plasmid to give the empty vector pYSZL32 ( AfpyrG-cyfp ) or pYSZL33 ( hph - nyfp ) using the Clone Express® MultiS One Step Cloning Kit, respectively. Each gene of csdA and laeB was fused with gpdA and integrated into pYSZL32 or pYSZL33 to obtain pYSZL25 or pYSZL28, respectively. Above plasmids were verified by sequencing (SANGON BIOTECH Co. Ltd, CHINA). Fungal genetic manipulations The csdA or laeB gene in A. fumigatus was deleted according to the method described previously 68 . Briefly, the deletion cassette of csdA or laeB was transformed into A. fumigatus CEA17.2 to produce single mutant TYYJ14 or TYSZL12, respectively 33 . For the subcellular localization, the laeB-sfGFP-AfpyrG (7.1 kb) and csdA-mCherry-hph (6.9 kb) fragments were amplified from pYSZL11 and pYSZL13, respectively, and were transformed into A. fumigatus CEA17.2 and CEA17.1 to produce strains TYSZL17 and TYSZL18. Meanwhile, the laeB-sfGFP-AfpyrG and csdA-mcherry-hph fragments were transformed together into A. fumigatus CEA17.2 to obtain the co-localized strain TYSZL21. For the BiFC assays, each pair of constructed vectors (pYSZL28 and pYSZL32, pYSZL33 and pYSZL25, pYSZL28 and pYSZL25) were co-transformed to A. fumigatus CEA17.2 to generate strains TYSZL37, TYSZL36, TYSZL25, respectively. The same method was used to construct TYLYX3 (Δ fmqC-hph ), TYSZL79 (Δ fmqC-hph , Δ laeB-AfpyrG ) and TYSZL80 (Δ fmqC-hph , Δ csdA-AfpyrG ). All the above transformants were verified by diagnostic PCR. Phylogenetic analysis of CsdA, LaeB or Sfp-type PPTases The amino acid sequences of CsdA and LaeB in A. fumigatus were obtained by multi-alignment with CsdA from P. fici or LaeB from A. nidulans , respectively. CsdA, LaeB and PptA 42 in A. fumigatus were used as the query for a BLAST analysis at the NCBI website ( www.blast.ncbi.nlm.nih.gov/Blast.cgi ). Amino acid sequences of CsdA, LaeB and PptA homologues from 94, 192 and 344 species were downloaded from the NCBI database, aligned with MEGA7 software, and manually adjusted. Three phylogenetic trees were constructed by MEGA7 software, and clustering were performed by the neighbor-joining method, while the other parameters were default. The fungi from Basidiomycota or Mucoromycota were regarded as the outgroups of CsdA or LaeB phylogram. The species from plants were regarded as the outgroup of Sfp-type PPTases phylogram. The reliability of internal branch was evaluated with 1,000 bootstrap resampling. The phylogram was modified and optimized via the Interactive Tree of Life website (ITOL, http://itol.embl.de/ ). Fungal growth and metabolomics analysis Aspergillus fumigatus CEA17 and its mutants were activated and cultivated on GMM medium at 37°C for 3 days. The conidia were collected with 0.1% Tween-80 and counted by a hemocytometer. Lung tissues from 6-week-old Balb/c mice were homogenized in 0.165 M MOPS buffer and supplemented with 3% agar to obtain lung plate medium. Approximately 1000 conidia of each strain were point-incubated on solid RPMI 1640 and mouse lung plate medium at 37°C for 3 days, and radial growth was measured daily. Each experiment was conducted in at least three biological replicates. To analyze fungal secondary metabolites, 1×10 7 spores of A. fumigatus CEA17 and its mutants were inoculated into 20 mL liquid GMM medium at 25°C for 5 days with shaking at 200 rpm. Other clinical and environmental strains were incubated in liquid RPMI 1640 medium at 37°C for 5 days. The metabolites were extracted with 20 mL of ethyl acetate and evaporated under reduced pressure. The extracts were dissolved in 1 mL methanol (MeOH), and then analysed by LC-HRMS equipped with an ODS column (C18, 250 × 4.6 mm, Waters XTERRA®, 5 µm) with a flow rate of 1 mL/min. The MeOH and water with 0.1% (v/v) formic acid were used as the elution solvent, and linear gradient conditions were as follows: 10%-30% MeOH in 0–10 min, 30%-70% MeOH in 10–40 min, 70%-90% MeOH in 40–50 min, 100% MeOH in 50.1–60 min, and 10% MeOH in 60.1–65 min. Clinical and environmental strains were analysed with an Agilent 1200 LC/MSD SL (Santa-Clara, USA) with a flow rate of 1 mL/min. The acetonitrile and water with 0.1% (v/v) formic acid were used as the elution solvent, and linear gradient conditions were as follows: 5%-100% acetonitrile in 0–30 min, 100% acetonitrile in 30–35 min, 100%-5% acetonitrile in 35–35.1 min, and 5% acetonitrile in 35.1–40 min. The metabolomics of the drug treated strains were analysed by the same method. The mass spectrum data were collected and converted into a format containing retention time, m/z , and ion peak density, respectively. Differentially regulated metabolites were screened with |log 2 foldchange|>1 and -log 10 ( p -value) > 1.3 as the threshold. Animal model of A. fumigatus infection Virulence of A. fumigatus Cea17.1 (WT), Δ csdA , Δ laeB , Δ csdA C , Δ laeB C , Δ fmqC , Δ csdA Δ fmqC and Δ laeB Δ fmqC strains were assessed in a murine invasive aspergillosis model. Briefly, male Balb/c mice (BEIJING VITAL RIVER LABORATORY ANIMAL TECHNOLOGY Co., Ltd., CHINA) weighing about 19–21 g were immunosuppressed via administration of cyclophosphamide by separate intraperitoneal injections, one at 3 days (200 mg·kg − 1 of body weight) and the other at 1 day (200 mg·kg − 1 of body weight) before infection. The second treatment includes administration of cortisone acetate at a dose of 250 mg·kg − 1 by separate subcutaneous injection at 1 day before infection. Anesthetized mice (10 mice/fungal strain) were infected by nasal instillation of 20 µL of 1x10 8 conidia/mL (day 0) and monitored three times daily for 12 days’ post-infection. All surviving mice were sacrificed at day 12. Survival analysis was performed by Log-rank (Mantel-Cox) test. Fungal burden and histopathological analysis Lung tissues from mice infected with A. fumigatus Cea17.1 or its mutants for 3 days were removed and freeze-dried. Subsequently, the dry lung tissue was homogenized in a CTAB (Cetyl trimethylammonium bromide, Sigma) extraction buffer (100 mM Tris-HCl pH 7.5, 0.7 M NaCl, 10 mM EDTA, 1% CTAB, 1% β-mercaptoethanol) to extract total gDNA as described previously 73 . Briefly, the mixed samples were cracked at 65°C for 30 min and then mixed with 1 mL chloroform and centrifuged at 1500 ×g for 10 min. All sample supernatants were added to isopropyl alcohol in equal volume and mixed gently. After centrifugation at 1500 ×g for 10 min, the precipitate was washed with 70% ethanol and dissolved with distilled water. 1 ng/µL of A. fumigatus gDNA was continuously diluted twofold to obtain 12 different concentrations for a standard curve. The X axis is the log 2 value of the known standard concentrations, and Y axis is the Ct value of each standard. The above extracted gDNA samples were diluted to 20 ng/µL, and Afks1 was used as the internal gene for qPCR quantification. The contents of A. fumigatus and its mutants in lung tissues were obtained according to the standard curve. Lungs removed from mice infected for 3 days were fixed with 10% formalin, and subsequently embedded in paraffin. Subsequently, consecutive slices with 4–6 µm in thickness were obtained and stained with haematoxylin-eosin (HE) and periodic acid-schiff (PAS) for histopathological studies. The fungal burden and histopathological of infected murine lungs after 5 days of drug treatment were analyzed using the same method. Fluorescence detection and BiFC assays For subcellular localization of CsdA or LaeB, an appropriate number of spores of TYSZL17 ( laeB-sfGFP ), TYSZL18 ( csdA-mCherry ), or TYSZL21 ( laeB-sfGFP, csdA-mCherry ) was inoculated into liquid GMM medium at 37 ºC and shaken at 200 rpm for 8–10 h. The mycelia collected by centrifugation were fixed in 10% formalin for 30 min and washed with distilled water and stained by DAPI solution (final concentration: 10 µg/mL, BIOSHARP, CHINA) for 15 min. The fluorescent images were obtained with a Zeiss Axioplan 2 imaging system with the AxioCam MRm camera (Carl Zeiss Microscopy) and were processed with IMAGEJ2 software (NATIONAL INSTITUTES OF HEALTH). The in vivo interaction between CsdA and LaeB was confirmed by the NYFP- or CYFP- tagged BiFC strains. The fluorescent images were obtained and processed as described above. Expression and purification of proteins Escherichia coli BL21 cells transformed with pYSZL47 were incubated at 37 ºC in LB medium containing 50 µg·mL − 1 kanamycin until OD 600 = 0.6 to express GST-CsdA recombinant protein. Next, 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was used to induce protein expression at 16°C for 20 h. The cells collected by centrifugation were lysed (50 mM Tris, 150 mM NaCl, 1 mM DTT, pH 7.3) by freeze-thaw, and debris was removed. Recombinant GST-CsdA protein was purified with GST-tagged resin (BEYOTIME, CHINA) and eluted by elution buffer (50 mM Tris, 150 mM NaCl, 10 mM GSH, pH 8.0). The expression and purification of LaeB was as described above, but a different lysis buffer was used (50 mM NaH 2 PO 4 ·2H 2 O, 300 mM NaCl, 10 mM imidazole, pH 8), wash buffer (40 mM imidazole), elution buffer (100 mM imidazole) and Ni-NTA resin (QIAGEN, CA). Target proteins were detected and quantified by 12% SDS-PAGE and Nano-Drop C2000 (Thermo Fisher Scientific), respectively. Pull-down and western blotting The interaction between CsdA and LaeB was confirmed by pull-down assays in vitro 33 . Briefly, individual GST-CsdA or LaeB-His protein was incubated with Ni-NTA resin at 4 ºC for 4 h in binding buffer. Both samples were centrifuged for 1 min at 4°C and 800 ×g, and the supernatant of CsdA was added to the precipitate of LaeB and incubated at 4 ºC overnight. Next, the above mixture was centrifuged for 1 min at 4°C and 800 ×g, and the precipitation was washed three times through the wash buffer. The CsdA-LaeB-resin mixture was denatured by heating, separated on 7.5% SDS-PAGE, and then transferred to polyvinylidene fluoride (PVDF) membrane (PALL, USA). The CsdA and LaeB protein was detected with mouse monoclonal antibodies anti-GST (PROTEINTECH, 1:7000 dilution) and anti-His (PROTEINTECH, 1:10000 dilution), respectively. HRP Goat-Anti-Mouse IgG (PROTEINTECH, 1:5000 dilution) was used to hybridize with anti-His or GST antibodies, respectively, and target bands were detected by ECL (THERMO, MA, USA). Transcriptional analysis by RNA-seq and qRT-PCR Total RNAs from the mycelia of A. fumigatus CEA17.1, Δ csdA and Δ laeB strains were isolated using TriZol™ kit (TRANSGEN BIOTECH, CHINA), and then the quality of RNA was evaluated by Agilent 2100 bioanalyzer. Total RNA examples were sequenced on Illumina NovaSeq 6000 (Illumina, USA) at NOVOGENE BIOTECH Co. Ltd. Data quality was controlled by fastp (version 0.19.7) 74 based on sequencing error rate for a single base less than 1%. The clean reads were mapped to the reference sequence and visualized by HISAT2 75 or Integrative Genomics Viewer (IGV) software, respectively. A total of 11,463 unique transcripts were detected in A. fumigatus and its mutants. Gene expression levels were represented using normalized FPKM (fragments per kilobase of transcript per million mapped reads). The differentially expressed genes were identified with p value and log 2 foldchange/ratio between A. fumigatus and mutants by DESeq2 76 . Three replicates were performed for each strain. Total RNAs of A. fumigatus Cea17.1, Δ laeB and Δ csdA were reverse transcribed into cDNA with an Evo M-MLV Plus cDNA Synthesis kit (ACCURATE BIOTECH Co. Ltd, China) for quantitative real-time PCR (qRT-PCR) assays according to the manufacture’s protocol. Briefly, qRT-PCR was conducted using a KAPA SYBR FAST qPCR Kit (Kapa biosystems, USA). The reaction including 2 × KAPA SYBR FAST qPCR Master Mix, 0.2 µM forward/reverse primer, about 2 µg cDNA template was carried out at 95°C for 3 min, followed by 40 cycles of (95°C for 3 s, 60°C for 20 s, 72°C for 20 s). Each cDNA sample was performed in triplicate, and relative expression levels were calculated using the 2 −ΔΔCt method 72 . The relative expression of csdA , laeB , fmqC and pptA was determined as above. Structure prediction and virtual screening To screen for antifungal drugs, the three-dimensional structure of PptA containing 357 amino acids was predicted by AlphaFold2 77 . Meanwhile, 3,019 FDA-approved drugs were preprocessed into 1,787 ligands that could be used for docking by BatchVinaGUI software 78 , 79 . Molecular docking of PptA with ligands was performed by BatchVinaGUI software, and 1,280 ligands were successfully matched. The ligands were ranked according to the binding affinity of PptA with different small molecule configurations (Supplementary Table 5). Topological models and binding sites were visualized by the PyMOL2.5 software package. The default parameters of the tools were used. Antifungal testing of candidate drugs The conidia of A. fumigatus CEA17 were diluted to 1×10 6 with 0.1% Tween80, and 1 µL was point-incubated in GMM medium containing 8 top-ranking drugs (final concentration 200 µM), and incubated at 37°C in the dark for 4 days to plot the growth curve. Inhibition ratio was determined by comparing the growth of drug-treated samples after 4 days of incubation with that of DMSO-samples by measuring colony diameter. To determine the relative expression of target gene after drug treatment, 1×10 7 spores were inoculated in 10 mL liquid RPMI1640 medium containing 8 top-ranked drugs, and total RNA was extracted after 3 days of culture at 37°C for qRT-PCR assay. Quantification of the metabolized ionic products after drug treatment was performed by Agilent 1200 LC/MSD SL. The inhibition ratio of clinical strains was measured by the same method. At least two replicates were performed for each strain. Quantification of FqC The extraction of FqC was carried out according to the same method described above. Briefly, A. fumigatus was cultured on GMM medium at 37°C for 4 days and extracted by equal volume ethyl acetate, dried by vacuum, and then re-suspended with 1 mL MeOH for HPLC analysis. For the determination of FqC, FqC was separated on a Waters HPLC system (Waters e2695, Waters 2998, Photodiode Array Detector) using an ODS column (C18, 250 × 4.6 mm, Waters XTERRA®, 5 µm) with a flow rate of 1 mL/min. The methanol (A) and water with 0.1% (v/v) formic acid (D) was used as the solvent. Elution conditions were as follows: 0 to 30 min, 20–100% A; 30 to 35 min, 100% A; 35 to 35.1 min, 100–20% A. UV absorptions at 254 nm were illustrated. The peaks of FqC in the crude extracts were determined according to the retention time and molecular weight of the standard. Subsequently, the relative production of FqC was calculated according to its peak area by normalized to the control group. At least two replicates were performed for each strain. Broad-spectrum activity determination of drug candidates To evaluate the broad-spectrum activity of tepotinib, ibrutinib, and eltrombopag, the human pathogens A. flavus , Mucor circinelloides , Cryptococcus neoforman s, C. gattii , and the plant pathogens Fusarium oxysporum were all tested. The 1,000 spores of A. flavus were incubated in GMM medium containing 200 µM drugs, and the inhibition ratio were calculated after 3 days, and the metabolic ion products were analysed after 4 days of dark culture at 37°C. The mycelia of M. circinelloides and F. oxysporum were incubated in PDA medium containing 200 µM drugs, and the inhibition ratio were calculated after 2 days or 4 days, and the metabolic ion products were analysed after 3 days or 5 days of dark culture at 28°C. The C. neoforman s and C. gattii were cultured in YPD medium to OD 600 = 0.1 at 30°C with shaking at 220 rpm, then diluted 1,000 times and incubated with drugs in a 96-well plate. The inhibition ratio was calculated after 1 day and the metabolic ionic products were detected after 2 days of incubation. At least two replicates were performed for each strain. Evaluation of the combined antifungal efficiency between candidate drugs and amphotericin B Amphotericin B was prepared into a solution with a concentration of 20 mM and then successively diluted into GMM plates with final concentrations of 0, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, 51.2, 100, 200 µM, respectively. The 1,000 spores of A. fumigatus were point-incubated in GMM medium and cultured at 37°C for 3 days without light. The colony diameter was measured daily and the inhibition ratio for 3 days was calculated. Subsequently, 200 µM tepotinib was mixed with 0, 0.4, 0.8, 1.6, 3.2, 6.4 µM amphotericin B to form GMM medium. A. fumigatus was cultured in GMM medium for 3 days to calculate the inhibition ratio, and the secondary metabolites were analysed after 4 days. The antifungal efficacy evaluation of ibrutinib and eltrombopag combined with amphotericin B was the same as above. Two replicates were performed for each strain. Quantification and statistical analysis Statistical parameters are shown in the corresponding Figure legends. All statistical analyses were done in GraphPad Prism8 software. Quantification data are generally presented as bar/line plots, with the error bar representing mean ± SD. Asterisks were used to indicate statistical significance, *stands for p < 0.05; ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Declarations Reporting Summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Data availability The data supporting the findings of the present study are available within the paper and its Supplementary Information. Code availability This paper does not report original code. Acknowledgments We thank Drs. Wenzhao Wang and Weixin Ke (Institute of Microbiology, Chinese Academy of Sciences) for their advice in metabolome data collection and animal infection. We thank Professor Zhonghua Ma from Zhejiang University for providing the bimolecular fluorescent complementary vectors. We thank Professor Cunwei Cao from Guangxi Medical University for providing clinical isolates of A. fumigatus . This work was supported by the Strategic Priority Research Program of Chinese Academy of Sciences [grant no. XDB0830000]; the National Natural Science Foundation of China [grant no. 32470046 and 32170066]; the Key Research Program of Frontier Sciences, Chinese Academy of Sciences [grant no. ZDBS-LY-SM016]; the Chinese Academy of Sciences Project for Young Scientists in Basic Research [grant no. YSBR-111]. Author contributions W.-B.Y. conceived the research and supervised the study. Z.S. performed fungal phenotypic analysis, HPLC analysis, transcriptome and metabolome analysis, phylogenetic analysis, subcellular localization, pull-down assay, BiFC assay, structural prediction, molecular docking and murine experiments. 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Genomic context of azole resistance mutations in Aspergillus fumigatus determined using whole-genome sequencing. mBio 6 , e00536-00515 (2015). Etienne, K. A. et al. Genomic diversity of azole-resistant Aspergillus fumigatus in the United States. mBio 12 , e0180321-0180321 (2021). Milshteyn, A., Colosimo, D. A. & Brady, S. F. Accessing bioactive natural products from the human microbiome. Cell Host Microbe 23 , 725-736 (2018). Wu, J. et al. The intestinal fungus Aspergillus tubingensis promotes polycystic ovary syndrome through a secondary metabolite. Cell Host Microbe 33 , 119-136.e111 (2025). Wang, G. et al. Fungal-fungal cocultivation leads to widespread secondary metabolite alteration requiring the partial loss-of-function VeA1 protein. Sci. Adv. 8 , eabo6094 (2022). Yin, W. B. et al. A nonribosomal peptide synthetase-derived iron(III) complex from the pathogenic fungus Aspergillus fumigatus . J. Am. Chem. Soc. 135 , 2064-2067 (2013). Harvey, C. J. B. et al. HEx: A heterologous expression platform for the discovery of fungal natural products. Sci. Adv. 4 , eaar5459 (2018). Wu, G. W. et al. Polyketide production of pestaloficiols and macrodiolide ficiolides revealed by manipulations of epigenetic regulators in an endophytic fungus. Org. Lett. 18 , 1832-1835 (2016). Bok, J. W. & Keller, N. P. Fast and easy method for construction of plasmid vectors using modified quick-change mutagenesis. Methods Mol. Biol. 944 , 163-174 (2012). Zhou, S. et al. A new regulator RsdA mediating fungal secondary metabolism has a detrimental impact on asexual development in Pestalotiopsis fici . Environ. Microbiol. 21 , 11 (2019). Jung, K. W. et al. Systematic functional profiling of transcription factor networks in Cryptococcus neoformans . Nat. Commun. 6 , 6757 (2015). Goldstein, L. D. et al. Prediction and quantification of splice events from RNA-seq data. PLoS One 11 , e0156132 (2016). Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5 , 621-628 (2008). Love, M. L., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15 , 550 (2014). Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596 , 583-589 (2021). Che, X., Liu, Q. & Zhang, L. An accurate and universal protein-small molecule batch docking solution using Autodock Vina. Results Eng. 19 , 101335 (2023). Morris, G. M. et al. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 30 , 2785-2791 (2009). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTable1.Massspectrometrydata.xlsx Dataset 1 SupplementaryTable3.Highresolutionmassspectrometrydata.xlsx Dataset 3 SupplementaryTable4.RNAseqdata.xlsx Dataset 4 SupplementaryTable5.AffinitydataforFDAapproveddrugwithtarget.xlsx Dataset 5 SupplementaryTable6.Massspectrometrydataofpathogenicfungi.xlsx Mass spectrometry data of pathogenic fungi SupplementaryTable7.Strainsplasmidsandprimers.xlsx Strains, plasmids and primers 2025CsdAlaeBSupplementaryFigures.pdf Supplementary Figures ExtendedDataFigures.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6615529","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":469888401,"identity":"8f09153e-ee1a-46ad-9520-bb63fc6fda1b","order_by":0,"name":"Wenbing 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Cai","email":"","orcid":"https://orcid.org/0000-0002-8131-7274","institution":"State Key Laboratory of Microbial Diversity and Innovative Utilization, Institute of Microbiology, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Cai","suffix":""},{"id":469888415,"identity":"4d60782b-dc3e-408b-96dc-cc7bb5f6b695","order_by":14,"name":"Koon Ho Wong","email":"","orcid":"https://orcid.org/0000-0002-9264-5118","institution":"University of Macau","correspondingAuthor":false,"prefix":"","firstName":"Koon","middleName":"Ho","lastName":"Wong","suffix":""}],"badges":[],"createdAt":"2025-05-08 01:20:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6615529/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6615529/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84447166,"identity":"f72609dc-521a-45e2-a390-2fa4f315c111","added_by":"auto","created_at":"2025-06-12 06:02:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":695590,"visible":true,"origin":"","legend":"\u003cp\u003eRNA binding protein CsdA governs global metabolism and pathogenicity of \u003cem\u003eA. fumigatus\u003c/em\u003e. a, Comparative metabolomics analysis of clinical and environmental \u003cem\u003eA. fumigatus\u003c/em\u003e. b, Differential metabolite production in clinical \u003cem\u003eA. fumigatus\u003c/em\u003e compared to environmental strain 34. c, Volcano plots showing the differentially regulated metabolic ion products in the Δ\u003cem\u003ecsdA\u003c/em\u003e mutant versus the control CEA17. d, Schematic diagram of growth and metabolome analysis of Δ\u003cem\u003ecsdA \u003c/em\u003emutant cultured in RPMI1640 and lung plate medium. e, Comparative analysis of Δ\u003cem\u003ecsdA\u003c/em\u003e mutant growth rate in RPMI1640 and lung plate medium. f, Differential metabolic ion products of Δ\u003cem\u003ecsdA \u003c/em\u003emutant in RPMI1640 and lung plate medium. g, Workflow for evaluating the virulence of Δ\u003cem\u003ecsdA\u003c/em\u003e mutant in the invasive aspergillosis model. h, Survival curves of Balb/c mice intranasally infected with \u003cem\u003ecsdA\u003c/em\u003e deletion and complementation mutants compared with control strains (\u003cem\u003en \u003c/em\u003e=10, log-rank test). i, Macroscopic pathology of the lung collected on day 3 post-infected by \u003cem\u003eA. fumigatus \u003c/em\u003eand its mutant. j, Analysis of \u003cem\u003eafks1\u003c/em\u003eexpression in the lungs of\u003cem\u003e A. fumigatus\u003c/em\u003eand its mutant after 3 days of infection (\u003cem\u003en\u003c/em\u003e =5). The expression level of \u003cem\u003eafks1\u003c/em\u003e was used to indicate fungal burden. k-l, Periodic Acid-Schiff (k) and Haematoxylin \u0026amp; Eosin (l) staining on day 3 post infection with \u003cem\u003eA. fumigatus\u003c/em\u003e and its mutant. Scale bars, 100 μm (×20, top panels), 50 μm (×40, bottom panels), and 25 μm (×80, bottom panels). All error bars are expressed as ± SD. Statistical analysis was performed by using Two-way ANOVA (Significant at *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6615529/v1/c4c65212426d622770e44825.png"},{"id":84447165,"identity":"ee695098-55a1-4472-9838-0a41a0e842b4","added_by":"auto","created_at":"2025-06-12 06:02:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":527566,"visible":true,"origin":"","legend":"\u003cp\u003eCsdA interacts with LaeB to orchestrate global metabolism and pathogenicity. a, Phylogenetic analysis of LaeB in pathogenic fungi. b, Target identification workflow of CsdA regulating secondary metabolism and virulence. c, Volcano plots showing the differentially regulated metabolic ion products in the Δ\u003cem\u003elaeB\u003c/em\u003e mutant versus the control. |Log\u003csub\u003e2\u003c/sub\u003efoldchange| \u0026gt; 1 and -Log\u003csub\u003e10\u003c/sub\u003e(\u003cem\u003ep\u003c/em\u003e-value) \u0026gt; 1.3 indicate a significant difference. d, Survival curves of Balb/c mice intranasally infected with \u003cem\u003elaeB\u003c/em\u003e deletion and complementation mutants compared with control strains (\u003cem\u003en \u003c/em\u003e=10, log-rank test). e, Periodic Acid-Schiff (top row) and Haematoxylin \u0026amp; Eosin (low row) staining on day 3 post-infection with \u003cem\u003eA. fumigatus\u003c/em\u003e and its mutant. Scale bars, 50 μm (×40, bottom panels), (yellow arrowheads indicate \u003cem\u003eA. fumigatu\u003c/em\u003es hyphae). f, Analysis of \u003cem\u003eafks1\u003c/em\u003e expression in the lungs of\u003cem\u003e A. fumigatus\u003c/em\u003e and its mutant after 3 days of infection (\u003cem\u003en\u003c/em\u003e =5). The expression level of \u003cem\u003eafks1\u003c/em\u003e indicated fungal burden. g, Co-localization of LaeB and CsdA in \u003cem\u003eA. fumigatus\u003c/em\u003e. LaeB-sfGFP and CsdA-mCherry localize in the nucleus and co-localize with DAPI signals. Nuclei in hyphae were stained with 4’, 6-diamidino-2-phenylindole (DAPI). h, The interaction between CsdA and LaeB was studied by \u003cem\u003ein vivo\u003c/em\u003e bimolecular fluorescence complementation (BiFC). The strains harboring a single construct (LaeB-YFP\u003csup\u003eN\u003c/sup\u003e or CsdA-YFP\u003csup\u003eC\u003c/sup\u003e) were used as negative controls. BF, bright-field. Scale bars, 5 μm. All error bars are expressed as ± SD. Statistical analysis was performed by using Two-way ANOVA.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6615529/v1/151192df4e8e2190b54cb270.png"},{"id":84445911,"identity":"6e754b79-5538-457b-9f29-8d09186ee3a9","added_by":"auto","created_at":"2025-06-12 05:37:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":626039,"visible":true,"origin":"","legend":"\u003cp\u003eCsdA or LaeB mediated global changes of secondary metabolism. a, Integrated transcriptomic-metabolomic analysis framework of CsdA- or LaeB-mediated global secondary metabolic change in \u003cem\u003eA. fumigatus\u003c/em\u003e. b, Transcriptomic and metabolomic analysis of genes and total metabolic ion products co-regulated by CsdA and LaeB in \u003cem\u003eA. fumigatus\u003c/em\u003e. c, GO enrichment analysis of genes co-regulated by CsdA and LaeB in \u003cem\u003eA. fumigatus\u003c/em\u003e. d, The gene expression levels of 39 BGC genes regulated by CsdA or LaeB in \u003cem\u003eA. fumigatus\u003c/em\u003e. Red and blue dots indicate the genes that are significantly up-regulated and down-regulated by LaeB, respectively. The size of the dots represents the genes that are regulated by CsdA. e, Ranking of BGCs regulated by CsdA or LaeB based on the expression intensity of the backbone genes in \u003cem\u003eA. fumigatus\u003c/em\u003e. f, The distribution of BGCs regulated by CsdA and LaeB alone or together in \u003cem\u003eA. fumigatus\u003c/em\u003e. g-h, Representative secondary metabolites FqC (g) and gliotoxin (h) co-regulated by CsdA and LaeB in \u003cem\u003eA. fumigatus\u003c/em\u003e. SebA and RglT are out-of-cluster transcription factors of\u003cem\u003e fmq\u003c/em\u003e and \u003cem\u003egli\u003c/em\u003e clusters, respectively. All error bars are expressed as ±SD. Statistical analysis was performed by using Two-way ANOVA (“ns”: not significant. Significant at *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6615529/v1/4293de9d27b7824a8d8fdc14.png"},{"id":84445912,"identity":"251fcbf3-aefc-4fce-a664-d5765f9667b8","added_by":"auto","created_at":"2025-06-12 05:37:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":666179,"visible":true,"origin":"","legend":"\u003cp\u003eMining FDA-approved drugs by targeting Sfp-type PPTases for antifungal therapy. a, Workflow of drugs screening and evaluation against\u003cem\u003eA. fumigatus\u003c/em\u003e. SMs: secondary metabolites. PptA: Sfp-type 4’-phosphopantetheinyl transferase (PPTase) in \u003cem\u003eA. fumigatus\u003c/em\u003e. b, Activity evaluation of the top eight drugs with high affinity to PptA against \u003cem\u003eA. fumigatus\u003c/em\u003e. c, Inhibition ratio analysis of three drug candidates with anti-\u003cem\u003eA. fumigatus\u003c/em\u003e activity. d, Structures of interaction between three drug candidates and the target PptA. Yellow and cyan represent PptA protein and drug structures, respectively. e, Expression analysis of the target \u003cem\u003epptA\u003c/em\u003e under drugs treatment by qRT-PCR assays. f, Metabolomics analysis of metabolic ion products inhibited by drugs in \u003cem\u003eA. fumigatus \u003c/em\u003eCEA17. g, Evaluation of FqC production in \u003cem\u003eA. fumigatus\u003c/em\u003e treated with three drug candidates. h, Activity evaluation of three candidate drugs against clinical \u003cem\u003eA. fumigatus\u003c/em\u003e. i, Relative abundance of FqC in clinical \u003cem\u003eA. fumigatus\u003c/em\u003e treated with three drug candidates. j, Relative abundance of FqC in each clinical \u003cem\u003eA. fumigatus\u003c/em\u003e treated with three drug candidates. k, Pulmonary fungal burden in a mouse model of invasive aspergillosis after 5 days of treatment with three drug candidates (\u003cem\u003en\u003c/em\u003e = 3). l, Periodic Acid-Schiff staining of \u003cem\u003eA. fumigatus\u003c/em\u003e in the mouse lungs after 5 days of treatment with three drug candidates. Scale bars: 25 μm (black arrowheads indicate \u003cem\u003eA. fumigatus\u003c/em\u003ehyphae). All error bars are expressed as ± SD. Statistical analysis was performed by using Two-way ANOVA (“ns”: not significant. Significant at *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6615529/v1/fd1bce30f4ac3a4fd1fbb825.png"},{"id":84445939,"identity":"5c0624f6-9df0-43b2-9374-0bf34e20f9ae","added_by":"auto","created_at":"2025-06-12 05:37:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":434793,"visible":true,"origin":"","legend":"\u003cp\u003eTargeted drugs exhibit broad-spectrum antifungal activities. a, Phylogenetic analysis of the target Sfp-type PPTases in fungi. PPTases: 4’-phosphopantetheinyl transferases. b, Conservative analysis of the binding sites in the pathogenic fungal Sfp-type PPTases. Binding sites D159, W227, E231, and K235 of the three drug candidates were conserved in fungal Sfp-type PPTases, including \u003cem\u003eA. fumigatus\u003c/em\u003e, \u003cem\u003eA. flavus\u003c/em\u003e, \u003cem\u003eMucor circinelloides\u003c/em\u003e, \u003cem\u003eFusarium oxysporum\u003c/em\u003e, \u003cem\u003eCryptococcus neoformans\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand\u003cem\u003e C. gattii\u003c/em\u003e. c-e, Evaluation of growth and metabolism of filamentous fungi \u003cem\u003eA. flavus \u003c/em\u003e(c), \u003cem\u003eM. circinelloides \u003c/em\u003e(d) and \u003cem\u003eF. oxysporum \u003c/em\u003e(e) treated by three drug candidates. f-g, Evaluation of growth (f) and metabolism (g) of the \u003cem\u003eC. neoformans\u003c/em\u003e and\u003cem\u003e C. gattii\u003c/em\u003e treated by three drug candidates. h, The metabolic profiles of \u003cem\u003eC. neoformans\u003c/em\u003e and \u003cem\u003eC. gattii\u003c/em\u003e after treatment by eltrombopag. i-j, Antifungal activity evaluation of three drug candidates alone (i) or in combination (j) with amphotericin B. Tep, Ibr, and Elt stand for tepotinib, ibrutinib, and eltrombopag, respectively. IC\u003csub\u003e50\u003c/sub\u003e, half maximal inhibitory concentration. AmB, amphotericin B. All error bars are expressed as ±SD. Inhibited metabolic ionic products: Log\u003csub\u003e2\u003c/sub\u003efoldchange \u0026lt; -1.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6615529/v1/8e784ad6e97097c2b81b2111.png"},{"id":85869952,"identity":"47c15cd8-382d-4102-bc3e-111825204829","added_by":"auto","created_at":"2025-07-02 13:58:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4447498,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6615529/v1/fcca9cc8-9d27-456f-a1bf-e98867a4673d.pdf"},{"id":84445940,"identity":"cc1f9c1d-b551-45de-b3e5-a7967854f85d","added_by":"auto","created_at":"2025-06-12 05:37:58","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":163765,"visible":true,"origin":"","legend":"Dataset 1","description":"","filename":"SupplementaryTable1.Massspectrometrydata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6615529/v1/de1d1ddc49baf2af9ac57c47.xlsx"},{"id":84445934,"identity":"68ec36e1-f361-4ff4-b7cb-7c8fddcf8bbb","added_by":"auto","created_at":"2025-06-12 05:37:57","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2375095,"visible":true,"origin":"","legend":"Dataset 3","description":"","filename":"SupplementaryTable3.Highresolutionmassspectrometrydata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6615529/v1/e9f89751c5c0f21587011a8e.xlsx"},{"id":84445930,"identity":"7f5a77d5-8643-4774-9e55-def4489abf82","added_by":"auto","created_at":"2025-06-12 05:37:57","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":139721,"visible":true,"origin":"","legend":"Dataset 4","description":"","filename":"SupplementaryTable4.RNAseqdata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6615529/v1/060e5c1b4bfb25ed872e751f.xlsx"},{"id":84445913,"identity":"d564a88d-be4d-4cb7-b60c-00ad73ed5946","added_by":"auto","created_at":"2025-06-12 05:37:57","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":531906,"visible":true,"origin":"","legend":"Dataset 5","description":"","filename":"SupplementaryTable5.AffinitydataforFDAapproveddrugwithtarget.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6615529/v1/b18719be5fa7181542d519b3.xlsx"},{"id":84445951,"identity":"a948e6d3-bac1-45c7-bcc5-0162f8a48aa6","added_by":"auto","created_at":"2025-06-12 05:37:58","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":8634482,"visible":true,"origin":"","legend":"Mass spectrometry data of pathogenic fungi","description":"","filename":"SupplementaryTable6.Massspectrometrydataofpathogenicfungi.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6615529/v1/8c9770a66f3ca109aa5aa2c3.xlsx"},{"id":84445921,"identity":"b871f135-d846-4b58-bbc8-bdbac6f3ccd3","added_by":"auto","created_at":"2025-06-12 05:37:57","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":27845,"visible":true,"origin":"","legend":"Strains, plasmids and primers","description":"","filename":"SupplementaryTable7.Strainsplasmidsandprimers.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6615529/v1/879afff6aa9bb419c6492ee1.xlsx"},{"id":84447164,"identity":"f12a11d6-e6f3-485c-bf26-ea7e53dd8fad","added_by":"auto","created_at":"2025-06-12 06:02:04","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":4418877,"visible":true,"origin":"","legend":"Supplementary Figures","description":"","filename":"2025CsdAlaeBSupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6615529/v1/c20a42e8590c6f6768eced21.pdf"},{"id":84447169,"identity":"79346610-bd0c-482f-8088-e8ffd0b5679c","added_by":"auto","created_at":"2025-06-12 06:02:56","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":8361005,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6615529/v1/88b71534dfe99c101e03486e.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"\u003cp\u003eCsdA-LaeB hub governs \u003cem\u003eAspergillus fumigatus\u003c/em\u003e virulence \u003cem\u003evia\u003c/em\u003e FqC biosynthesis\u003c/p\u003e","fulltext":[{"header":"Main","content":"\u003cp\u003eInvasive aspergillosis, a life-threatening fungal infection primarily caused by the opportunistic pathogen \u003cem\u003eAspergillus fumigatus\u003c/em\u003e, represents a significant global health burden and remains a leading cause of mortality \u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Annually, over 2\u0026nbsp;million individuals develop invasive aspergillosis, primarily in the context of chronic obstructive pulmonary disease (COPD), intensive care, lung cancer, or hematological malignancies, resulting in an estimated crude annual mortality of 1.8\u0026nbsp;million \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eA. fumigatus\u003c/em\u003e, a formidable human pathogen, predominantly infects immunocompromised individuals, such as cancer patients, transplant recipients, and those grappling with COPD \u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. To successfully invade the host, \u003cem\u003eA. fumigatus\u003c/em\u003e employs a range of virulence strategies, including thermotolerance, cell wall modifications, nutritional adaptability, stress responses, and interactions with the host immune system \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Notably, during host-pathogen interactions, bioactive secondary metabolites (SMs) produced by the invading \u003cem\u003eA. fumigatus\u003c/em\u003e play a crucial role in modulating immune responses \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Examples include DHN-melanin, which confers resistance to reactive oxygen species (ROS), and gliotoxin, which impairs immune cell function \u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, the regulatory networks governing their biosynthesis during infection, as well as their potential as therapeutic targets, remain poorly understood.\u003c/p\u003e \u003cp\u003eFungal SMs are encoded by biosynthetic gene clusters (BGCs). In \u003cem\u003eA. fumigatus\u003c/em\u003e, 20 BGCs have been linked to specific SMs \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. SM production is regulated by complex hierarchical networks, that include pathway-specific, epigenetic, and global regulation \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Several pathway-specific transcription factors (e.g., GliZ for gliotoxin), epigenetic modifiers (e.g., histone deacetylase RpdA) and global regulators (e.g., LaeA, control approximately 50% BGCs) have been characterized \u003csup\u003e\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. RNA-binding proteins (RBPs) emerge as a novel class of regulators influencing eukaryotic gene expression, are well studied in human disease\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e and plant development\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, yet their roles in fungal metabolism and virulence are veiled in an enigma. Furthermore, although global regulators such as LaeB are known to coordinate BGC activation \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, their interplay with RBPs and relevance to clinical isolates remain uncharted.\u003c/p\u003e \u003cp\u003eHere, we bridge this gap by integrating pan-metabolomics, functional genetics, and drug repurposing to unravel a novel regulatory hub driving \u003cem\u003eA. fumigatus\u003c/em\u003e virulence. Through pan-metabolome analysis, we identified distinct metabolic profiles between clinical and environmental \u003cem\u003eA. fumigatus\u003c/em\u003e isolates, with fumiquinazoline C (FqC) emerging as a major contributor to virulence. Transcriptomic and metabolomic analyses revealed an unknown interaction between CsdA and LaeB, confirmed by \u003cem\u003ein vitro\u003c/em\u003e pull-down and \u003cem\u003ein vivo\u003c/em\u003e bimolecular fluorescence complementation assays. Strikingly, disruption of this complex hyperactivated FqC production, increasing fungal colonization and virulence, revealing a delicate balance between metabolic regulation and host adaptation. Importantly, we identified Sfp-type PPTases \u0026mdash; a conserved class of phosphopantetheinyl transferases critical for widespread SMs biosynthesis \u0026mdash; as a universal target for anti-infective therapy. We further demonstrated that tepotinib, ibrutinib, and eltrombopag inhibit FqC synthesis, and consequently reducing fungal colonization and inflammation in mouse lungs. This study provides an insightful perspective on the metabolic regulation of fungal pathogenesis and proposes a novel anti-infective strategy targeting global secondary metabolism.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eMetabolic divergence between clinical and environmental\u003c/b\u003e \u003cb\u003eA. fumigatus\u003c/b\u003e \u003cb\u003eisolates\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo explore the link between \u003cem\u003eA. fumigatus\u003c/em\u003e metabolism and virulence, we performed a non-targeted pan-metabolomic analysis of 19 \u003cem\u003eA. fumigatus\u003c/em\u003e isolates, including 5 environmental and 13 clinical strains, as well as model strain CEA17 (as also clinical isolate) (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Table\u0026nbsp;1). Total ion peaks give a good indication of the relative number of metabolites produced. Principal components analysis (PCA) revealed significant metabolic differences between clinical and environmental isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). To quantify the metabolic differences between the two groups, we compared the abundance of each detected ion across isolates using the environmental isolate 34 as a reference. Quantitative profiling revealed 11%-12% of detected metabolic ion products exhibited abundance variations in all other environmental isolates. Strikingly, clinical isolates demonstrated substantially greater divergence, with 25%-64% of metabolic ion products differing from environmental isolate 34, including a 61% variation in the model strain CEA17 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). These findings suggest a link between global metabolism and \u003cem\u003eA. fumigatus\u003c/em\u003e virulence.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCsdA governs global metabolism and virulence\u003c/h2\u003e \u003cp\u003eFungal metabolism is usually controlled by a series of key regulatory proteins. We recently discovered an RNA-binding protein (RBP) CsdA in the endophytic fungus \u003cem\u003ePestalotiopsis fici\u003c/em\u003e that is critical for normal growth and metabolism \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. RBPs play crucial roles in regulating pathogenicity in various eukaryotes, yet their mechanisms of action in fungal pathogenicity remain unclear. Analysis of fungal genome data revealed CsdA homologues existing in fungal kingdom especially in pathogenic fungi (Supplementary Fig.\u0026nbsp;1), including \u003cem\u003eAspergillus spp.\u003c/em\u003e, \u003cem\u003eHistoplasma capsulatum\u003c/em\u003e, \u003cem\u003eBlastomyces gilchristii\u003c/em\u003e, and dermatophytes (\u003cem\u003eArthroderma uncinatum, Trichophyton rubrum, T. mentagrophytes\u003c/em\u003e), \u003cem\u003eCandida auris\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e. Interestingly, the identity/coverage of CsdA homology in common fungal pathogen \u003cem\u003eB. gilchristii\u003c/em\u003e was 83.76%/100% relative to the \u003cem\u003eA. fumigatus\u003c/em\u003e homolog. Basidiomycete fungi (\u003cem\u003eCryptococcus neoformans var. grubii\u003c/em\u003e) exhibited lower identity/coverage (30%/10%) but retained similar RNA recognition motif (RRM) and zinc finger (ZnF) domains (Supplementary Fig.\u0026nbsp;1 and Supplementary Table\u0026nbsp;2). This suggests that CsdA is prevalent in Ascomycota, and notably in fungal pathogens.\u003c/p\u003e \u003cp\u003eTo examine the function of CsdA, a comparative metabolome analysis between the Δ\u003cem\u003ecsdA\u003c/em\u003e mutant and control CEA17 strain revealed that 1,321 (26% of total) ion peaks were significantly regulated among the detected 5,057 products (adjusted \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log\u003csub\u003e2\u003c/sub\u003efoldchange|\u0026gt;1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and Supplementary Table\u0026nbsp;3). To mimic the host\u0026rsquo;s lung environment, we compared the growth and metabolite profiles of Δ\u003cem\u003ecsdA\u003c/em\u003e mutant between RPMI1640 medium (a commonly used cell medium) and lung plate (agar supplemented mouse lung homogenate) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). This mutant showed a faster growth in the lung plate compared to the RPMI1640 medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Through a comparative metabolomics analysis, we found that the metabolic ion products regulated by CsdA in RPMI1640 medium and lung plate were significantly higher than GMM medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, f). Among them, 3,252 (75% of total) ion peaks in RPMI1640 medium were regulated by CsdA, including 1,510 (35%) down-regulated and 1,742 (40%) up-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Similarly, 3,042 (78% of total) ion peaks in lung plate were regulated by CsdA, including 1,593 (41%) down-regulated and 1,449 (37%) up-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eGiven the established role of \u003cem\u003eA. fumigatus\u003c/em\u003e toxins in virulence \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, we presumed that metabolites regulated by CsdA might impact the virulence of this pathogen. In a neutropenic murine model of invasive aspergillosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg), infection with the Δ\u003cem\u003ecsdA\u003c/em\u003e mutant resulted in 100% mortality by day 8, compared to 70% in the control strain. Complementation of \u003cem\u003ecsdA\u003c/em\u003e restored mortality to wild-type levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). Meanwhile, the Δ\u003cem\u003ecsdA\u003c/em\u003e mutant also had a higher fungal burden in infected murine lungs, concomitant with the higher mortality (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei, j). Histological examination revealed extensive lung tissue damage in Δ\u003cem\u003ecsdA\u003c/em\u003e-infected mice, characterized by the extensive conidial germination and invasive mycelium formation. In contrast, the control strain only exhibited limited pulmonary epithelial invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek, l).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCsdA interacts with LaeB to orchestrate metabolism and virulence\u003c/h3\u003e\n\u003cp\u003ePreviously, we demonstrated that CsdA interacts with RsdA to regulate secondary metabolism in \u003cem\u003eP. fici\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Upon systematic investigation, RsdA\u0026rsquo;s ortholog protein with 49%/80% identity/coverage was found in \u003cem\u003eA. fumigatus\u003c/em\u003e. Interestingly, the protein is also identified in other \u003cem\u003eAspergillus\u003c/em\u003e species including \u003cem\u003eA. flavus\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eA. nidulans\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, known as LaeB (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and Supplementary Fig.\u0026nbsp;2). In these species, LaeB, a protein with undefined domains, has an established role in regulating secondary metabolism, although its function in \u003cem\u003eA. fumigatus\u003c/em\u003e remains uninvestigated. Thus, a Δ\u003cem\u003elaeB\u003c/em\u003e mutant was constructed in the CEA17 background to characterize its function (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Similar to the Δ\u003cem\u003ecsdA\u003c/em\u003e mutant, a comparative metabolomics analysis revealed a total of 5,045 ion peaks were detected, among which 2,083 (41%) displayed significant changes in the Δ\u003cem\u003elaeB\u003c/em\u003e mutant (adjusted \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log\u003csub\u003e2\u003c/sub\u003efoldchange|\u0026gt;1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and Supplementary Table\u0026nbsp;3). Notably, 1,575 (31%) were significantly down-regulated, while 508 (10%) were significantly up-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). In the neutropenic murine model, we observed that the Δ\u003cem\u003elaeB\u003c/em\u003e mutant \u0026ndash; similar to the Δ\u003cem\u003ecsdA\u003c/em\u003e mutant \u0026ndash; also displayed a 100% fatality rate at 7 days after infection, and the \u003cem\u003elaeB\u003c/em\u003e complement strain was restored to near WT levels of virulence (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Histological analysis of the murine lungs infected with the Δ\u003cem\u003elaeB\u003c/em\u003e mutant also exhibited severe pathology characterized by invasive mycelium, along with a significantly heightened fungal burden when compared to the control strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, f). This suggests a fascinating connection of LaeB and CsdA in mediating fungal virulence.\u003c/p\u003e \u003cp\u003eTo probe the relationship between CsdA and LaeB, we analysed the subcellular localization of CsdA and LaeB in \u003cem\u003eA. fumigatus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Fluorescent labelling of both proteins (LaeB-sfGFP and CsdA-mCherry) coupled with DAPI (4\u0026rsquo;,6-diamidino-2-phenylindole, a DNA binding dye) staining revealed that CsdA and LaeB co-localize in the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Next, we expressed LaeB-His and GST-CsdA fusions in bacteria and used them in pull-down assay. GST-CsdA (92.4 kDa) was co-purified when LaeB-His (89.6 kDa) was pulled down using anti-His antibody, suggesting a direct physical interaction between LaeB and CsdA (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d). To confirm the interaction between the two proteins in the nucleus, we created \u003cem\u003elaeB-YFP\u003c/em\u003e\u003csup\u003e\u003cem\u003eN\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ecsdA-YFP\u003c/em\u003e\u003csup\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sup\u003e fusions in \u003cem\u003eA. fumigatus\u003c/em\u003e and used them in a bimolecular fluorescence complementation (BiFC) assay. We observed a yellow fluorescent signal indicating that the two proteins interact physically and the signal co-localized with DAPI indicating that the interaction happens in the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). Together, these results demonstrate that CsdA and LaeB interact to form a functional complex.\u003c/p\u003e\n\u003ch3\u003eCsdA/LaeB regulates key secondary metabolites, including FqC\u003c/h3\u003e\n\u003cp\u003eTo investigate how loss of either CsdA or LaeB increases virulence, we constructed a regulatory network co-mediated by both proteins, involving co-regulated metabolic ions and genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Venn analysis identified 809 metabolic ions with significantly altered abundances in both Δ\u003cem\u003ecsdA\u003c/em\u003e and Δ\u003cem\u003elaeB\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Transcriptomic analysis revealed that 2,380 (21%) and 2,789 (24%) genes exhibited over twofold higher expression in the Δ\u003cem\u003ecsdA\u003c/em\u003e and Δ\u003cem\u003elaeB\u003c/em\u003e mutants, respectively, compared to the control strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and Supplementary Table\u0026nbsp;4). A total of 911 genes were significantly regulated in both mutants, predominantly related to secondary metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Since most secondary metabolites (SMs) are encoded by biosynthetic gene clusters (BGCs), we mapped gene expression profiles of all 39 BGCs in the \u003cem\u003eA. fumigatus\u003c/em\u003e genome \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Of these, 15 (39%) backbone genes were significantly co-regulated in both mutants, with 13 displaying the same expression trend, including 3 up-regulated and 10 down-regulated, such as \u003cem\u003efmqC\u003c/em\u003e, \u003cem\u003eencA\u003c/em\u003e, \u003cem\u003egliP\u003c/em\u003e, \u003cem\u003eafumA\u003c/em\u003e, \u003cem\u003enscA\u003c/em\u003e, \u003cem\u003eetc\u003c/em\u003e. (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-d).\u003c/p\u003e \u003cp\u003eIn addition to gliotoxin \u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eA. fumigatus\u003c/em\u003e produces multiple mycotoxins \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e that modulate host immunity, such as endocrocin \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e and fumiquinazoline C (FqC) \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. To explore how CsdA and LaeB regulate these specific mycotoxins, we systematically analysed the BGCs controlled by the two proteins and categorized them based on the expression intensity of their backbone genes. Among the most prominently affected BGCs upon \u003cem\u003ecsdA\u003c/em\u003e deletion, gliotoxin, fumiquinazoline, and endocrocin biosynthesis genes were up-regulated, while fumihopaside and neosartoricin biosynthesis genes were significantly down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, f).\u003c/p\u003e \u003cp\u003eSpecifically, fumiquinazoline production involves five genes (\u003cem\u003efmqA-E\u003c/em\u003e), with four of them (\u003cem\u003efmqA-D\u003c/em\u003e) negatively regulated by the \u003cem\u003etrans\u003c/em\u003e-acting transcription factor SebA \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In both Δ\u003cem\u003ecsdA\u003c/em\u003e and Δ\u003cem\u003elaeB\u003c/em\u003e mutants, \u003cem\u003efmqB\u003c/em\u003e-\u003cem\u003eD\u003c/em\u003e were significantly up-regulated, while \u003cem\u003esebA\u003c/em\u003e was significantly down-regulated. Metabolomics analysis revealed a dramatic 19.84- and 13.35-fold increase of FqC production in the Δ\u003cem\u003ecsdA\u003c/em\u003e or Δ\u003cem\u003elaeB\u003c/em\u003e mutants, respectively, compared to the control strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). This suggested that CsdA and LaeB co-regulate the expression of \u003cem\u003efmqB-D\u003c/em\u003e through SebA, and ultimately influencing FqC production. In contrast, the immunosuppressant gliotoxin production was only subtly up-regulated by 2.11- and 2.77-fold in the Δ\u003cem\u003ecsdA\u003c/em\u003e and Δ\u003cem\u003elaeB\u003c/em\u003e mutants, respectively, compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). The biosynthesis of gliotoxin involves 13 genes within the cluster and one transcription factor RglT outside the cluster \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In both mutants, the expression of \u003cem\u003egliM\u003c/em\u003e and \u003cem\u003egliK\u003c/em\u003e, along with 12 other genes (includin \u003cem\u003egliZ\u003c/em\u003e and \u003cem\u003erglT\u003c/em\u003e), was up-regulated to varying degrees (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). Based on current understanding of the endocrocin biosynthetic pathway \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, four genes (\u003cem\u003eencA-D\u003c/em\u003e) in the cluster are involved. Transcriptome analysis revealed the upregulation of four genes by more than twofold in both mutants, with LaeB exerting more significant regulation than CsdA (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Conversely, all gene expressions in the neosartoricin and fumihopaside BGCs were significantly down-regulated in both mutants (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, g). Collectively, although CsdA/LaeB orchestrates multiple mycotoxins, FqC emerges as the predominant regulated metabolite.\u003c/p\u003e\n\u003ch3\u003eFqC is a key virulence metabolite\u003c/h3\u003e\n\u003cp\u003eTo further gain additional support for the regulation of secondary metabolism contributing to virulence in the \u003cem\u003ecsdA\u003c/em\u003e and \u003cem\u003elaeB\u003c/em\u003e deletion mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), we measured the expression levels of \u003cem\u003ecsdA\u003c/em\u003e and \u003cem\u003elaeB\u003c/em\u003e as well as their co-regulated BGC backbone genes by qRT-PCR across clinical strains. The results showed that \u003cem\u003ecsdA\u003c/em\u003e and \u003cem\u003elaeB\u003c/em\u003e expression were significantly down-regulated in clinical strains compared to the control strain CEA17, with \u003cem\u003elaeB\u003c/em\u003e exhibiting greater repression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c). Interestingly, the backbone gene expression and production of FqC were significantly up-regulated across multiple clinical strains compared to the control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), suggesting a potential role for this metabolite in \u003cem\u003eA. fumigatus\u003c/em\u003e pathogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, e).\u003c/p\u003e \u003cp\u003eWe simulated the culture of clinical strains in mouse lung homogenates to identify the key pathogenic metabolites. Compared with control CEA17, FqC production in other clinical strains were up-regulated by more than twofold (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results further support the role of FqC in \u003cem\u003eA. fumigatus\u003c/em\u003e pathogenicity. Subsequently, a Δ\u003cem\u003efmqC\u003c/em\u003e mutant was constructed in the same CEA17 background (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), and its virulence compared to the Δ\u003cem\u003ecsdA\u003c/em\u003e mutant and wild-type strains. The Δ\u003cem\u003efmqC\u003c/em\u003e mutant exhibited significantly attenuated virulence, with 60% mortality at 8 days\u0026rsquo; post-infection compared to 90% in wild-type strain (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). Quantitative fungal burden analysis revealed reduced lung colonization in mice infected with Δ\u003cem\u003efmqC\u003c/em\u003e mutant compared with wild-type strains (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). These findings suggest that CsdA-mediated pathogenesis may be primarily attributable to its regulation on FqC biosynthesis.\u003c/p\u003e \u003cp\u003eTo explore whether FqC serves as the key metabolite underlying CsdA- and LaeB-induced virulence, we generated Δ\u003cem\u003ecsdA\u003c/em\u003eΔ\u003cem\u003efmqC\u003c/em\u003e and Δ\u003cem\u003elaeB\u003c/em\u003eΔ\u003cem\u003efmqC\u003c/em\u003e double mutants in the CEA17 background (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) and compared their virulence to the Δ\u003cem\u003efmqC\u003c/em\u003e single mutant and wild-type strains. Metabolic profiling revealed that neither the Δ\u003cem\u003ecsdA\u003c/em\u003eΔ\u003cem\u003efmqC\u003c/em\u003e and Δ\u003cem\u003elaeB\u003c/em\u003eΔ\u003cem\u003efmqC\u003c/em\u003e double mutants nor the Δ\u003cem\u003efmqC\u003c/em\u003e single mutant produced FqC, compared to the control strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). Critically, the mortality of Δ\u003cem\u003ecsdA\u003c/em\u003eΔ\u003cem\u003efmqC\u003c/em\u003e and Δ\u003cem\u003elaeB\u003c/em\u003eΔ\u003cem\u003efmqC\u003c/em\u003e double mutants was restored to wild-type levels, compared to that of the single mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej). Furthermore, there were no significant differences in fungal colonization or histopathology between the double mutants and the control strains (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek, l). These findings establish FqC as the key effector of CsdA/LaeB-mediated pathogenicity.\u003c/p\u003e\n\u003ch3\u003eTargeting Sfp-type PPTase for antifungal therapy\u003c/h3\u003e\n\u003cp\u003eMultiple SMs contributes to the overall \u003cem\u003eA. fumigatus\u003c/em\u003e pathogenicity \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. To dismantle \u003cem\u003eA. fumigatus\u003c/em\u003e pathogenicity at its metabolic core, we focused on Sfp-type PPTase\u0026mdash;a 4\u0026rsquo;-phosphopantetheinyl transferase essential for synthesizing polyketides (PKs) and non-ribosomal peptides (NRPs), two major virulence-associated SM classes, known as PptA in \u003cem\u003eA. fumigatus\u003c/em\u003e \u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Strikingly, deletion of \u003cem\u003epptA\u003c/em\u003e in the CEA17 background revealed near-complete disruption of hyphal growth, asexual development, and global secondary metabolism, demonstrating its potential as a potent antifungal target (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-d).\u003c/p\u003e \u003cp\u003eSubsequently, we predicted PptA structure using Alphafold2, and performed virtual screening of 3,019 marketed drugs in the FDA-Approved Drug Library (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and Supplementary Fig.\u0026nbsp;3). According to the affinity between PptA and ligands, all candidate drugs were scored and ranked, and the antifungal activities of the top eight drugs were evaluated \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, Supplementary Figs.\u0026nbsp;4,5 and Supplementary Table\u0026nbsp;5). Three drugs\u0026mdash;tepotinib, ibrutinib, and eltrombopag\u0026mdash;demonstrated significant growth inhibition against \u003cem\u003eA. fumigatus\u003c/em\u003e, with inhibition ratios of 67%, 27%, and 36%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-d). To validate PptA targeting, we performed qRT-PCR assay, revealing 2.8-, 5.2-, and 4.9-fold reductions in \u003cem\u003epptA\u003c/em\u003e expression following three drug treatments (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Meanwhile, we also analysed the metabolic ionic products of the drug-treated CEA17 strain (Supplementary Table\u0026nbsp;6). Crucially, they broadly compromised SM production, reducing FqC levels by 1.3\u0026ndash;2.2-fold (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and globally suppressing 32\u0026ndash;53% of metabolic ion products (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef, g, Extended Data Figs.\u0026nbsp;7\u0026ndash;9 and Supplementary Fig.\u0026nbsp;6). The therapeutic potential of this strategy was validated across 13 clinical \u003cem\u003eA. fumigatus\u003c/em\u003e isolates, with all three tested drugs demonstrating varying degrees of growth and metabolic inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh-j). Notably, tepotinib exhibited the most potent activity, achieving up to 70% growth inhibition and reducing FqC production by up to 6.3-fold (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In murine models of invasive aspergillosis, intranasal discontinuous administration for 5 days reduced pulmonary fungal burden by 1.7 to 2.1-fold (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and attenuated tissue damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek, l), demonstrating \u003cem\u003ein vivo\u003c/em\u003e efficacy against \u003cem\u003eA. fumigatus\u003c/em\u003e infection. These findings reveal targeting secondary metabolic biosynthetic pathway as a promising therapeutic strategy for combating invasive aspergillosis.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBroad-spectrum antifungal activity of drug candidates\u003c/h2\u003e \u003cp\u003eGiven the ubiquitous presence of SMs across fungi, we conducted a comprehensive phylogenetic analysis of PptA orthologs (Supplementary Fig.\u0026nbsp;7). Evolutionary conservation analysis revealed that PptA orthologs are widely distributed among fungi, with four critical drug-binding sites (D159, W227, E231, and K235) showing remarkable conservation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee and Supplementary Fig.\u0026nbsp;8), suggesting potential broad-spectrum antifungal activity of drug candidates. Then, we evaluated the efficacy of tepotinib, ibrutinib, and eltrombopag against divergent fungi (\u003cem\u003eA. flavus\u003c/em\u003e, \u003cem\u003eMucor circinelloides\u003c/em\u003e, \u003cem\u003eFusarium oxysporum\u003c/em\u003e, \u003cem\u003eC. neoformans, C. gattii\u003c/em\u003e). Tepotinib emerged as the most potent agent against \u003cem\u003eA. flavus\u003c/em\u003e, inhibiting growth by 57% and metabolites production by 52% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec and Supplementary Fig.\u0026nbsp;9). Ibrutinib showed maximal efficacy against \u003cem\u003eF. oxysporum\u003c/em\u003e and \u003cem\u003eM. circinelloides\u003c/em\u003e, achieving 73% and 68% growth suppression, as well as 37% and 42% metabolic inhibition (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, e, Supplementary Fig.\u0026nbsp;9), respectively. Strikingly, eltrombopag demonstrated maximal efficacy against \u003cem\u003eCryptococcus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef), and significantly inhibiting metabolism in both \u003cem\u003eC. neoformans\u003c/em\u003e H99 (64% reduction) and \u003cem\u003eC. gattii\u003c/em\u003e R265 (59% reduction) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, h).\u003c/p\u003e \u003cp\u003eTo enhance clinical relevance, we combined these agents with amphotericin B (AmB), a cell membrane-targeting fungicide \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Compared to standalone IC\u003csub\u003e50\u003c/sub\u003e values of AmB (0.92 \u0026micro;M), tepotinib, ibrutinib, and eltrombopag showed higher concentration (100.4\u0026ndash;765.9 \u0026micro;M) against \u003cem\u003eA. fumigatus\u003c/em\u003e. Strikingly, combination with AmB significantly enhanced efficacy (0.12\u0026ndash;0.35 \u0026micro;M), with combination index of 0.14\u0026ndash;0.38 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei, j and Extended Data Fig.\u0026nbsp;10). This dual-target strategy\u0026mdash;simultaneously disrupting metabolic virulence and cell integrity\u0026mdash;represents a paradigm shift in circumventing antifungal resistance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough \u003cem\u003eA. fumigatus\u003c/em\u003e pathogenicity has been characterized at genomic and transcriptomic levels \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, the contribution of global secondary metabolism to infection remains enigmatic \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Fungal pathogenicity has traditionally been attributed to thermotolerance \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, cell wall modifications \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, nutritional adaptability \u003csup\u003e\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, biofilm \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, and stress responses \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. However, our findings reveal an additional layer of pathogenesis: the remodeling of secondary metabolism to coordinate virulence. Here, we identify the CsdA-LaeB complex as a master conductor of this process, bridging SM dynamics to virulence. Integrative metabolomic profiling of clinical versus environmental isolates uncovered FqC as a clinically enriched SM that is critical for lung invasion and establishment of invasive aspergillosis.\u003c/p\u003e \u003cp\u003eSince the biosynthetic genes of fumiquinazoline were identified in 2010 \u003csup\u003e57\u003c/sup\u003e, its role in virulence remained unknown until this study. Pan-metabolomics analysis of clinical and environmental isolates of \u003cem\u003eA. fumigatus\u003c/em\u003e demonstrated differences in metabolic ion abundance between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). Notably, FqC was significantly enriched in clinical strains and negatively regulated by CsdA, suggesting its involvement in the CsdA-mediated regulatory pathway controlling of \u003cem\u003eA. fumigatus\u003c/em\u003e virulence (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-f). This was subsequently demonstrated by constructing double mutants of Δ\u003cem\u003efmqC\u003c/em\u003eΔ\u003cem\u003ecsdA\u003c/em\u003e and Δ\u003cem\u003efmqC\u003c/em\u003eΔ\u003cem\u003elaeB\u003c/em\u003e and in a murine model of invasive aspergillosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej-l). Genetic dissection revealed that CsdA and its nuclear interactor LaeB that orchestrates FqC biosynthesis and virulence in \u003cem\u003eA. fumigatus\u003c/em\u003e by forming a regulatory hub CsdA-LaeB-FqC. Disruption of the complex (Δ\u003cem\u003ecsdA\u003c/em\u003e or Δ\u003cem\u003elaeB\u003c/em\u003e) triggered a 13.4\u0026ndash;19.8-fold FqC surge (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg), correlating with a rise in murine mortality (up to 100% by day 7 post-infection, Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed)\u0026mdash;a phenomenon implies evolutionary trade-offs between metabolic dynamics and virulence.\u003c/p\u003e \u003cp\u003eFungal RBPs frequently function within protein complexes to regulate cellular processes \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Distinct from the functions of regulating growth and development reported in \u003cem\u003eF. graminearum\u003c/em\u003e (FgRbp1-U2AF23) \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eM. oryzae\u003c/em\u003e (RBP35-CFI25) \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, this discovery positions RBP complex as central players in fungal metabolism and virulence. Metabolomics and transcriptomic analysis showed that 809 metabolic ion products and 911 genes (including 33% BGC backbone genes) were co-regulated by CsdA and LaeB (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). This is consistent with the regulatory mode of secondary metabolism in the endophytic fungus \u003cem\u003eP. fici\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Current efforts to distinguish clinical \u003cem\u003eA. fumigatus\u003c/em\u003e strains rely on pan-genomics \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e or azole-resistance markers \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e, overlooking metabolic drivers. Our pan-metabolomic profiling of clinical versus environmental isolates identified FqC as a potential diagnostic biomarker and therapeutic target. By targeting PptA\u0026mdash;a conserved phosphopantetheinyl transferase essential for widespread SMs biosynthesis (including FqC)\u0026mdash;we repurposed three FDA-approved drugs (tepotinib, ibrutinib, eltrombopag) to cripple fungal infection. These drugs inhibited \u003cem\u003eA. fumigatus\u003c/em\u003e growth by 27\u0026ndash;67% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), suppressed FqC production by 1.3\u0026ndash;2.2-fold (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and reduced pulmonary fungal burden in invasive aspergillosis mouse models by 1.7\u0026ndash;2.1-fold (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Therapeutic efficacy was validated across 13 clinical \u003cem\u003eA. fumigatus\u003c/em\u003e isolates, with all three drugs showing dose-dependent antifungal effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh-j). Strikingly, tepotinib demonstrated superior potency, achieving 70% growth inhibition and a 6.3-fold reduction in FqC production (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). These findings establish PptA as a key controller of SM biosynthesis, including FqC production, and highlight targeting secondary metabolic biosynthetic pathway as a promising therapeutic strategy for combating invasive aspergillosis.\u003c/p\u003e \u003cp\u003eWhile the scientific community is often fixated on the dual - edged nature of SMs, marveling at their roles as either potent natural drugs\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e or menacing virulence factors\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e, an entire realm of untapped potential lies hidden: the remarkable promise of their biosynthetic pathways as powerful anti-infection targets. The species-specific biosynthesis of SMs in fungi\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, underscores their potential as an innovative, highly selective and safe therapeutic target. The high conservation of drug-binding sites (D159, W227, E231, K235) across fungal Sfp-type PPTases highlights its potential as a broad-spectrum antifungal target (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). Among the tested pathogens, tepotinib was the most potent against \u003cem\u003eA. flavus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec), ibrutinib showed maximal efficacy against \u003cem\u003eF. oxysporum\u003c/em\u003e and \u003cem\u003eM. circinelloides\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, e). Remarkably, eltrombopag exhibited maximal efficacy against \u003cem\u003eCryptococcus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef), and significantly inhibiting metabolism in both \u003cem\u003eC. neoformans\u003c/em\u003e H99 and \u003cem\u003eC. gattii\u003c/em\u003e R265 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, h), which may aid in combating fatal fungal infection. Furthermore, three agents also demonstrated potent synergistic effects with amphotericin B, revealing a novel dual-target antifungal strategy that combines conventional cell membrane disruption with secondary metabolism inhibition. These antifungal effects targeting non-traditional targets, combined with dual-target synergy (AmB\u0026thinsp;+\u0026thinsp;PptA inhibitors), establishes a blueprint for combating multidrug-resistant infections.\u003c/p\u003e \u003cp\u003eIn conclusion, our work redefines antifungal therapy by shifting focus from traditional drug target of cell membrane to metabolic choke points. The CsdA-LaeB-FqC hub illuminates how pathogens dynamically recalibrate virulence metabolites, while Sfp-type PPTase-targeted drug repurposing delivers an actionable strategy to outmaneuver resistance. By deciphering pathogen\u0026rsquo;s \u0026ldquo;metabolic virulence code\u0026rdquo;, we provide both a mechanistic framework and a therapeutic toolkit to combat the escalating threat of invasive fungal diseases.\u003c/p\u003e "},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies\u003c/h2\u003e \u003cp\u003eAnti-His, Anti-GST and HRP-Goat Anti-Mouse from Proteintech (66005-1-Ig, 66001-2-Ig, SA00001-1) were used for western blot assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003eAll murine experiments were performed in strict accordance with the \u0026ldquo;the regulation of the Institute of Microbiology, Chinese Academy of Sciences of Research Ethics Committee.\u0026rdquo; The murine experiment protocol was approved by the Institute of Microbiology, Chinese Academy of Sciences of Research Ethics Committee (Permit No. APIMCAS2022106).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eBalb/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Male mice (age, 7\u0026ndash;8 weeks) were injected with cyclophosphamide and cortisone acetate to generate a murine model of invasive aspergillosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStrains and cultivation\u003c/h2\u003e \u003cp\u003eThe strains used in this study are listed in key resources Supplementary Table\u0026nbsp;7. All \u003cem\u003eAspergillus fumigatus\u003c/em\u003e strains were grown at 37\u0026deg;C on glucose minimum medium (GMM) with appropriate supplements corresponding to the auxotrophic marker or antibiotics \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eA. fumigatus\u003c/em\u003e and its transformants were cultivated in liquid GMM medium at 25\u0026deg;C for 3 days to extract total RNAs and for 5 days to detect secondary metabolites (SMs). \u003cem\u003eEscherichia coli\u003c/em\u003e strains DH5α and BL21 were cultured in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl) supplemented with appropriate antibiotics to construct plasmids and express proteins, respectively. \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e BJ5464 \u003csup\u003e69\u003c/sup\u003e were used for construction of green fluorescent protein (sfGFP) and red fluorescent protein (mCherry) expression vectors on synthetic dextrose complete medium with appropriate supplements corresponding to the auxotrophic markers \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGene cloning and plasmid construction\u003c/h2\u003e \u003cp\u003eAll plasmids and primers (BEIJING TSINGKE BIOTECH Co., Ltd., CHINA) are given in Supplementary Table\u0026nbsp;7. For the construction of deletion mutants, around 1 kb upstream and downstream fragments of the targeted genes were amplified from \u003cem\u003eA. fumigatus\u003c/em\u003e genomic DNA (gDNA) by high-fidelity DNA polymerase TransStart \u0026reg; FastPfu (TRANSGEN BIOTECH, CHINA). The marker genes \u003cem\u003eAfpyrG\u003c/em\u003e and \u003cem\u003ehph\u003c/em\u003e were amplified from the vectors pYH-WA-pyrG and pXW55-hph, respectively, and fused with the flanking sequences of the target genes to form different deletion cassettes by the double-joint PCR method described previously \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. For protein expression, high-fidelity DNA polymerase Q5 (NEW ENGLAND BIOLABS) was used to amplify the open reading frames (ORFs) of targeted genes that were then inserted into pET28a (\u003cem\u003eHis\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e-tag) or pGEX-4T (\u003cem\u003eGST\u003c/em\u003e-tag) to produce pYSZL46 (LaeB-His) or pYSZL47 (GST-CsdA) through the quick-change method \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. For the subcellular localization of CsdA, the \u003cem\u003ecsdA\u003c/em\u003e, \u003cem\u003emCherry\u003c/em\u003e, and \u003cem\u003ehph\u003c/em\u003e were integrated into the \u003cem\u003eSpe\u003c/em\u003eI/\u003cem\u003ePml\u003c/em\u003eI-cleaved pXW55 vector \u003cem\u003evia\u003c/em\u003e the yeast recombination method \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e to give the plasmid pYSZL13 (\u003cem\u003ecsdA-mCherry\u003c/em\u003e). The same method was used to construct \u003cem\u003esfGFP\u003c/em\u003e expression vectors pYSZL11 (\u003cem\u003elaeB-sfGFP\u003c/em\u003e). In order to construct complementary vectors, \u003cem\u003ecsdA\u003c/em\u003e and \u003cem\u003ehph\u003c/em\u003e were integrated into the \u003cem\u003eSpe\u003c/em\u003eI/\u003cem\u003ePml\u003c/em\u003eI-cleaved pXW55 vector \u003cem\u003evia\u003c/em\u003e the Clone Express\u0026reg; MultiS One Step Cloning Kit (VAZYME BIOTECH Co. Ltd, CHINA) to give the plasmid pYSZL56 (\u003cem\u003ecsdA-hph\u003c/em\u003e). The same method was used to construct pYSZL57 (\u003cem\u003elaeB-hph\u003c/em\u003e). The above plasmids were verified by PCR with 2\u0026times;GS Taq polymerase (GENESAND, CHINA).\u003c/p\u003e \u003cp\u003eTo construct \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ebi\u003c/span\u003emolecular \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ef\u003c/span\u003eluorescence \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ec\u003c/span\u003eomplementation (BiFC) vectors, the \u003cem\u003eAfpyrG\u003c/em\u003e or \u003cem\u003ehph\u003c/em\u003e gene was cloned into the \u003cem\u003eKpn\u003c/em\u003eI/\u003cem\u003eHind\u003c/em\u003eIII-cleaved pCX62-CYFP or pKNT-NYFP plasmid to give the empty vector pYSZL32 (\u003cem\u003eAfpyrG-cyfp\u003c/em\u003e) or pYSZL33 (\u003cem\u003ehph\u003c/em\u003e-\u003cem\u003enyfp\u003c/em\u003e) using the Clone Express\u0026reg; MultiS One Step Cloning Kit, respectively. Each gene of \u003cem\u003ecsdA\u003c/em\u003e and \u003cem\u003elaeB\u003c/em\u003e was fused with \u003cem\u003egpdA\u003c/em\u003e and integrated into pYSZL32 or pYSZL33 to obtain pYSZL25 or pYSZL28, respectively. Above plasmids were verified by sequencing (SANGON BIOTECH Co. Ltd, CHINA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFungal genetic manipulations\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ecsdA\u003c/em\u003e or \u003cem\u003elaeB\u003c/em\u003e gene in \u003cem\u003eA. fumigatus\u003c/em\u003e was deleted according to the method described previously \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Briefly, the deletion cassette of \u003cem\u003ecsdA\u003c/em\u003e or \u003cem\u003elaeB\u003c/em\u003e was transformed into \u003cem\u003eA. fumigatus\u003c/em\u003e CEA17.2 to produce single mutant TYYJ14 or TYSZL12, respectively \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. For the subcellular localization, the \u003cem\u003elaeB-sfGFP-AfpyrG\u003c/em\u003e (7.1 kb) and \u003cem\u003ecsdA-mCherry-hph\u003c/em\u003e (6.9 kb) fragments were amplified from pYSZL11 and pYSZL13, respectively, and were transformed into \u003cem\u003eA. fumigatus\u003c/em\u003e CEA17.2 and CEA17.1 to produce strains TYSZL17 and TYSZL18. Meanwhile, the \u003cem\u003elaeB-sfGFP-AfpyrG\u003c/em\u003e and \u003cem\u003ecsdA-mcherry-hph\u003c/em\u003e fragments were transformed together into \u003cem\u003eA. fumigatus\u003c/em\u003e CEA17.2 to obtain the co-localized strain TYSZL21. For the BiFC assays, each pair of constructed vectors (pYSZL28 and pYSZL32, pYSZL33 and pYSZL25, pYSZL28 and pYSZL25) were co-transformed to \u003cem\u003eA. fumigatus\u003c/em\u003e CEA17.2 to generate strains TYSZL37, TYSZL36, TYSZL25, respectively. The same method was used to construct TYLYX3 (Δ\u003cem\u003efmqC-hph\u003c/em\u003e), TYSZL79 (Δ\u003cem\u003efmqC-hph\u003c/em\u003e, Δ\u003cem\u003elaeB-AfpyrG\u003c/em\u003e) and TYSZL80 (Δ\u003cem\u003efmqC-hph\u003c/em\u003e, Δ\u003cem\u003ecsdA-AfpyrG\u003c/em\u003e). All the above transformants were verified by diagnostic PCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis of CsdA, LaeB or Sfp-type PPTases\u003c/h2\u003e \u003cp\u003eThe amino acid sequences of CsdA and LaeB in \u003cem\u003eA. fumigatus\u003c/em\u003e were obtained by multi-alignment with CsdA from \u003cem\u003eP. fici\u003c/em\u003e or LaeB from \u003cem\u003eA. nidulans\u003c/em\u003e, respectively. CsdA, LaeB and PptA \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e in \u003cem\u003eA. fumigatus\u003c/em\u003e were used as the query for a BLAST analysis at the NCBI website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003c/span\u003e). Amino acid sequences of CsdA, LaeB and PptA homologues from 94, 192 and 344 species were downloaded from the NCBI database, aligned with MEGA7 software, and manually adjusted. Three phylogenetic trees were constructed by MEGA7 software, and clustering were performed by the neighbor-joining method, while the other parameters were default. The fungi from Basidiomycota or Mucoromycota were regarded as the outgroups of CsdA or LaeB phylogram. The species from plants were regarded as the outgroup of Sfp-type PPTases phylogram. The reliability of internal branch was evaluated with 1,000 bootstrap resampling. The phylogram was modified and optimized \u003cem\u003evia\u003c/em\u003e the Interactive Tree of Life website (ITOL, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://itol.embl.de/\u003c/span\u003e\u003cspan address=\"http://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFungal growth and metabolomics analysis\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAspergillus fumigatus\u003c/em\u003e CEA17 and its mutants were activated and cultivated on GMM medium at 37\u0026deg;C for 3 days. The conidia were collected with 0.1% Tween-80 and counted by a hemocytometer. Lung tissues from 6-week-old Balb/c mice were homogenized in 0.165 M MOPS buffer and supplemented with 3% agar to obtain lung plate medium. Approximately 1000 conidia of each strain were point-incubated on solid RPMI 1640 and mouse lung plate medium at 37\u0026deg;C for 3 days, and radial growth was measured daily. Each experiment was conducted in at least three biological replicates.\u003c/p\u003e \u003cp\u003eTo analyze fungal secondary metabolites, 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e spores of \u003cem\u003eA. fumigatus\u003c/em\u003e CEA17 and its mutants were inoculated into 20 mL liquid GMM medium at 25\u0026deg;C for 5 days with shaking at 200 rpm. Other clinical and environmental strains were incubated in liquid RPMI 1640 medium at 37\u0026deg;C for 5 days. The metabolites were extracted with 20 mL of ethyl acetate and evaporated under reduced pressure. The extracts were dissolved in 1 mL methanol (MeOH), and then analysed by LC-HRMS equipped with an ODS column (C18, 250 \u0026times; 4.6 mm, Waters XTERRA\u0026reg;, 5 \u0026micro;m) with a flow rate of 1 mL/min. The MeOH and water with 0.1% (v/v) formic acid were used as the elution solvent, and linear gradient conditions were as follows: 10%-30% MeOH in 0\u0026ndash;10 min, 30%-70% MeOH in 10\u0026ndash;40 min, 70%-90% MeOH in 40\u0026ndash;50 min, 100% MeOH in 50.1\u0026ndash;60 min, and 10% MeOH in 60.1\u0026ndash;65 min. Clinical and environmental strains were analysed with an Agilent 1200 LC/MSD SL (Santa-Clara, USA) with a flow rate of 1 mL/min. The acetonitrile and water with 0.1% (v/v) formic acid were used as the elution solvent, and linear gradient conditions were as follows: 5%-100% acetonitrile in 0\u0026ndash;30 min, 100% acetonitrile in 30\u0026ndash;35 min, 100%-5% acetonitrile in 35\u0026ndash;35.1 min, and 5% acetonitrile in 35.1\u0026ndash;40 min. The metabolomics of the drug treated strains were analysed by the same method. The mass spectrum data were collected and converted into a format containing retention time, \u003cem\u003em/z\u003c/em\u003e, and ion peak density, respectively. Differentially regulated metabolites were screened with |log\u003csub\u003e2\u003c/sub\u003efoldchange|\u0026gt;1 and -log\u003csub\u003e10\u003c/sub\u003e (\u003cem\u003ep\u003c/em\u003e-value)\u0026thinsp;\u0026gt;\u0026thinsp;1.3 as the threshold.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnimal model of\u003c/b\u003e \u003cb\u003eA. fumigatus\u003c/b\u003e \u003cb\u003einfection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eVirulence of \u003cem\u003eA. fumigatus\u003c/em\u003e Cea17.1 (WT), Δ\u003cem\u003ecsdA\u003c/em\u003e, Δ\u003cem\u003elaeB\u003c/em\u003e, Δ\u003cem\u003ecsdA\u003c/em\u003e\u003csup\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sup\u003e, Δ\u003cem\u003elaeB\u003c/em\u003e\u003csup\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sup\u003e, Δ\u003cem\u003efmqC\u003c/em\u003e, Δ\u003cem\u003ecsdA\u003c/em\u003eΔ\u003cem\u003efmqC\u003c/em\u003e and Δ\u003cem\u003elaeB\u003c/em\u003eΔ\u003cem\u003efmqC\u003c/em\u003e strains were assessed in a murine invasive aspergillosis model. Briefly, male Balb/c mice (BEIJING VITAL RIVER LABORATORY ANIMAL TECHNOLOGY Co., Ltd., CHINA) weighing about 19\u0026ndash;21 g were immunosuppressed via administration of cyclophosphamide by separate intraperitoneal injections, one at 3 days (200 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of body weight) and the other at 1 day (200 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of body weight) before infection. The second treatment includes administration of cortisone acetate at a dose of 250 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e by separate subcutaneous injection at 1 day before infection. Anesthetized mice (10 mice/fungal strain) were infected by nasal instillation of 20 \u0026micro;L of 1x10\u003csup\u003e8\u003c/sup\u003e conidia/mL (day 0) and monitored three times daily for 12 days\u0026rsquo; post-infection. All surviving mice were sacrificed at day 12. Survival analysis was performed by Log-rank (Mantel-Cox) test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eFungal burden and histopathological analysis\u003c/h2\u003e \u003cp\u003eLung tissues from mice infected with \u003cem\u003eA. fumigatus\u003c/em\u003e Cea17.1 or its mutants for 3 days were removed and freeze-dried. Subsequently, the dry lung tissue was homogenized in a CTAB (Cetyl trimethylammonium bromide, Sigma) extraction buffer (100 mM Tris-HCl pH 7.5, 0.7 M NaCl, 10 mM EDTA, 1% CTAB, 1% β-mercaptoethanol) to extract total gDNA as described previously \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. Briefly, the mixed samples were cracked at 65\u0026deg;C for 30 min and then mixed with 1 mL chloroform and centrifuged at 1500 \u0026times;g for 10 min. All sample supernatants were added to isopropyl alcohol in equal volume and mixed gently. After centrifugation at 1500 \u0026times;g for 10 min, the precipitate was washed with 70% ethanol and dissolved with distilled water. 1 ng/\u0026micro;L of \u003cem\u003eA. fumigatus\u003c/em\u003e gDNA was continuously diluted twofold to obtain 12 different concentrations for a standard curve. The X axis is the log\u003csub\u003e2\u003c/sub\u003e value of the known standard concentrations, and Y axis is the Ct value of each standard. The above extracted gDNA samples were diluted to 20 ng/\u0026micro;L, and \u003cem\u003eAfks1\u003c/em\u003e was used as the internal gene for qPCR quantification. The contents of \u003cem\u003eA. fumigatus\u003c/em\u003e and its mutants in lung tissues were obtained according to the standard curve.\u003c/p\u003e \u003cp\u003eLungs removed from mice infected for 3 days were fixed with 10% formalin, and subsequently embedded in paraffin. Subsequently, consecutive slices with 4\u0026ndash;6 \u0026micro;m in thickness were obtained and stained with haematoxylin-eosin (HE) and periodic acid-schiff (PAS) for histopathological studies. The fungal burden and histopathological of infected murine lungs after 5 days of drug treatment were analyzed using the same method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence detection and BiFC assays\u003c/h2\u003e \u003cp\u003eFor subcellular localization of CsdA or LaeB, an appropriate number of spores of TYSZL17 (\u003cem\u003elaeB-sfGFP\u003c/em\u003e), TYSZL18 (\u003cem\u003ecsdA-mCherry\u003c/em\u003e), or TYSZL21 (\u003cem\u003elaeB-sfGFP, csdA-mCherry\u003c/em\u003e) was inoculated into liquid GMM medium at 37 \u0026ordm;C and shaken at 200 rpm for 8\u0026ndash;10 h. The mycelia collected by centrifugation were fixed in 10% formalin for 30 min and washed with distilled water and stained by DAPI solution (final concentration: 10 \u0026micro;g/mL, BIOSHARP, CHINA) for 15 min. The fluorescent images were obtained with a Zeiss Axioplan 2 imaging system with the AxioCam MRm camera (Carl Zeiss Microscopy) and were processed with IMAGEJ2 software (NATIONAL INSTITUTES OF HEALTH). The \u003cem\u003ein vivo\u003c/em\u003e interaction between CsdA and LaeB was confirmed by the NYFP- or CYFP- tagged BiFC strains. The fluorescent images were obtained and processed as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eExpression and purification of proteins\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e BL21 cells transformed with pYSZL47 were incubated at 37 \u0026ordm;C in LB medium containing 50 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e kanamycin until OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.6 to express GST-CsdA recombinant protein. Next, 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was used to induce protein expression at 16\u0026deg;C for 20 h. The cells collected by centrifugation were lysed (50 mM Tris, 150 mM NaCl, 1 mM DTT, pH 7.3) by freeze-thaw, and debris was removed. Recombinant GST-CsdA protein was purified with GST-tagged resin (BEYOTIME, CHINA) and eluted by elution buffer (50 mM Tris, 150 mM NaCl, 10 mM GSH, pH 8.0). The expression and purification of LaeB was as described above, but a different lysis buffer was used (50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, 300 mM NaCl, 10 mM imidazole, pH 8), wash buffer (40 mM imidazole), elution buffer (100 mM imidazole) and Ni-NTA resin (QIAGEN, CA). Target proteins were detected and quantified by 12% SDS-PAGE and Nano-Drop C2000 (Thermo Fisher Scientific), respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003ePull-down and western blotting\u003c/h2\u003e \u003cp\u003eThe interaction between CsdA and LaeB was confirmed by pull-down assays \u003cem\u003ein vitro\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Briefly, individual GST-CsdA or LaeB-His protein was incubated with Ni-NTA resin at 4 \u0026ordm;C for 4 h in binding buffer. Both samples were centrifuged for 1 min at 4\u0026deg;C and 800 \u0026times;g, and the supernatant of CsdA was added to the precipitate of LaeB and incubated at 4 \u0026ordm;C overnight. Next, the above mixture was centrifuged for 1 min at 4\u0026deg;C and 800 \u0026times;g, and the precipitation was washed three times through the wash buffer. The CsdA-LaeB-resin mixture was denatured by heating, separated on 7.5% SDS-PAGE, and then transferred to polyvinylidene fluoride (PVDF) membrane (PALL, USA). The CsdA and LaeB protein was detected with mouse monoclonal antibodies anti-GST (PROTEINTECH, 1:7000 dilution) and anti-His (PROTEINTECH, 1:10000 dilution), respectively. HRP Goat-Anti-Mouse IgG (PROTEINTECH, 1:5000 dilution) was used to hybridize with anti-His or GST antibodies, respectively, and target bands were detected by ECL (THERMO, MA, USA).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eTranscriptional analysis by RNA-seq and qRT-PCR\u003c/h2\u003e \u003cp\u003eTotal RNAs from the mycelia of \u003cem\u003eA. fumigatus\u003c/em\u003e CEA17.1, Δ\u003cem\u003ecsdA\u003c/em\u003e and Δ\u003cem\u003elaeB\u003c/em\u003e strains were isolated using TriZol\u0026trade; kit (TRANSGEN BIOTECH, CHINA), and then the quality of RNA was evaluated by Agilent 2100 bioanalyzer. Total RNA examples were sequenced on Illumina NovaSeq 6000 (Illumina, USA) at NOVOGENE BIOTECH Co. Ltd. Data quality was controlled by fastp (version 0.19.7)\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e based on sequencing error rate for a single base less than 1%. The clean reads were mapped to the reference sequence and visualized by HISAT2 \u003csup\u003e75\u003c/sup\u003e or Integrative Genomics Viewer (IGV) software, respectively. A total of 11,463 unique transcripts were detected in \u003cem\u003eA. fumigatus\u003c/em\u003e and its mutants. Gene expression levels were represented using normalized FPKM (fragments per kilobase of transcript per million mapped reads). The differentially expressed genes were identified with \u003cem\u003ep\u003c/em\u003e value and log\u003csub\u003e2\u003c/sub\u003efoldchange/ratio between \u003cem\u003eA. fumigatus\u003c/em\u003e and mutants by DESeq2 \u003csup\u003e76\u003c/sup\u003e. Three replicates were performed for each strain.\u003c/p\u003e \u003cp\u003eTotal RNAs of \u003cem\u003eA. fumigatus\u003c/em\u003e Cea17.1, Δ\u003cem\u003elaeB\u003c/em\u003e and Δ\u003cem\u003ecsdA\u003c/em\u003e were reverse transcribed into cDNA with an \u003cem\u003eEvo M-MLV\u003c/em\u003e Plus cDNA Synthesis kit (ACCURATE BIOTECH Co. Ltd, China) for quantitative real-time PCR (qRT-PCR) assays according to the manufacture\u0026rsquo;s protocol. Briefly, qRT-PCR was conducted using a KAPA SYBR FAST qPCR Kit (Kapa biosystems, USA). The reaction including 2 \u0026times; KAPA SYBR FAST qPCR Master Mix, 0.2 \u0026micro;M forward/reverse primer, about 2 \u0026micro;g cDNA template was carried out at 95\u0026deg;C for 3 min, followed by 40 cycles of (95\u0026deg;C for 3 s, 60\u0026deg;C for 20 s, 72\u0026deg;C for 20 s). Each cDNA sample was performed in triplicate, and relative expression levels were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. The relative expression of \u003cem\u003ecsdA\u003c/em\u003e, \u003cem\u003elaeB\u003c/em\u003e, \u003cem\u003efmqC\u003c/em\u003e and \u003cem\u003epptA\u003c/em\u003e was determined as above.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eStructure prediction and virtual screening\u003c/h2\u003e \u003cp\u003eTo screen for antifungal drugs, the three-dimensional structure of PptA containing 357 amino acids was predicted by AlphaFold2 \u003csup\u003e77\u003c/sup\u003e. Meanwhile, 3,019 FDA-approved drugs were preprocessed into 1,787 ligands that could be used for docking by BatchVinaGUI software \u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e,\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. Molecular docking of PptA with ligands was performed by BatchVinaGUI software, and 1,280 ligands were successfully matched. The ligands were ranked according to the binding affinity of PptA with different small molecule configurations (Supplementary Table\u0026nbsp;5). Topological models and binding sites were visualized by the PyMOL2.5 software package. The default parameters of the tools were used.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eAntifungal testing of candidate drugs\u003c/h2\u003e \u003cp\u003eThe conidia of \u003cem\u003eA. fumigatus\u003c/em\u003e CEA17 were diluted to 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e with 0.1% Tween80, and 1 \u0026micro;L was point-incubated in GMM medium containing 8 top-ranking drugs (final concentration 200 \u0026micro;M), and incubated at 37\u0026deg;C in the dark for 4 days to plot the growth curve. Inhibition ratio was determined by comparing the growth of drug-treated samples after 4 days of incubation with that of DMSO-samples by measuring colony diameter. To determine the relative expression of target gene after drug treatment, 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e spores were inoculated in 10 mL liquid RPMI1640 medium containing 8 top-ranked drugs, and total RNA was extracted after 3 days of culture at 37\u0026deg;C for qRT-PCR assay. Quantification of the metabolized ionic products after drug treatment was performed by Agilent 1200 LC/MSD SL. The inhibition ratio of clinical strains was measured by the same method. At least two replicates were performed for each strain.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eQuantification of FqC\u003c/h2\u003e \u003cp\u003eThe extraction of FqC was carried out according to the same method described above. Briefly, \u003cem\u003eA. fumigatus\u003c/em\u003e was cultured on GMM medium at 37\u0026deg;C for 4 days and extracted by equal volume ethyl acetate, dried by vacuum, and then re-suspended with 1 mL MeOH for HPLC analysis. For the determination of FqC, FqC was separated on a Waters HPLC system (Waters e2695, Waters 2998, Photodiode Array Detector) using an ODS column (C18, 250 \u0026times; 4.6 mm, Waters XTERRA\u0026reg;, 5 \u0026micro;m) with a flow rate of 1 mL/min. The methanol (A) and water with 0.1% (v/v) formic acid (D) was used as the solvent. Elution conditions were as follows: 0 to 30 min, 20\u0026ndash;100% A; 30 to 35 min, 100% A; 35 to 35.1 min, 100\u0026ndash;20% A. UV absorptions at 254 nm were illustrated. The peaks of FqC in the crude extracts were determined according to the retention time and molecular weight of the standard. Subsequently, the relative production of FqC was calculated according to its peak area by normalized to the control group. At least two replicates were performed for each strain.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eBroad-spectrum activity determination of drug candidates\u003c/h2\u003e \u003cp\u003eTo evaluate the broad-spectrum activity of tepotinib, ibrutinib, and eltrombopag, the human pathogens \u003cem\u003eA. flavus\u003c/em\u003e, \u003cem\u003eMucor circinelloides\u003c/em\u003e, \u003cem\u003eCryptococcus neoforman\u003c/em\u003es, \u003cem\u003eC. gattii\u003c/em\u003e, and the plant pathogens \u003cem\u003eFusarium oxysporum\u003c/em\u003e were all tested. The 1,000 spores of \u003cem\u003eA. flavus\u003c/em\u003e were incubated in GMM medium containing 200 \u0026micro;M drugs, and the inhibition ratio were calculated after 3 days, and the metabolic ion products were analysed after 4 days of dark culture at 37\u0026deg;C. The mycelia of \u003cem\u003eM. circinelloides\u003c/em\u003e and \u003cem\u003eF. oxysporum\u003c/em\u003e were incubated in PDA medium containing 200 \u0026micro;M drugs, and the inhibition ratio were calculated after 2 days or 4 days, and the metabolic ion products were analysed after 3 days or 5 days of dark culture at 28\u0026deg;C. The \u003cem\u003eC. neoforman\u003c/em\u003es and \u003cem\u003eC. gattii\u003c/em\u003e were cultured in YPD medium to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.1 at 30\u0026deg;C with shaking at 220 rpm, then diluted 1,000 times and incubated with drugs in a 96-well plate. The inhibition ratio was calculated after 1 day and the metabolic ionic products were detected after 2 days of incubation. At least two replicates were performed for each strain.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of the combined antifungal efficiency between candidate drugs and amphotericin B\u003c/h2\u003e \u003cp\u003eAmphotericin B was prepared into a solution with a concentration of 20 mM and then successively diluted into GMM plates with final concentrations of 0, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, 51.2, 100, 200 \u0026micro;M, respectively. The 1,000 spores of \u003cem\u003eA. fumigatus\u003c/em\u003e were point-incubated in GMM medium and cultured at 37\u0026deg;C for 3 days without light. The colony diameter was measured daily and the inhibition ratio for 3 days was calculated. Subsequently, 200 \u0026micro;M tepotinib was mixed with 0, 0.4, 0.8, 1.6, 3.2, 6.4 \u0026micro;M amphotericin B to form GMM medium. \u003cem\u003eA. fumigatus\u003c/em\u003e was cultured in GMM medium for 3 days to calculate the inhibition ratio, and the secondary metabolites were analysed after 4 days. The antifungal efficacy evaluation of ibrutinib and eltrombopag combined with amphotericin B was the same as above. Two replicates were performed for each strain.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eQuantification and statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical parameters are shown in the corresponding Figure legends. All statistical analyses were done in GraphPad Prism8 software. Quantification data are generally presented as bar/line plots, with the error bar representing mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Asterisks were used to indicate statistical significance, *stands for \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ****\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eReporting Summary\u003c/p\u003e\n\u003cp\u003eFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of the present study are available within the paper and its Supplementary Information.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCode availability\u003c/p\u003e\n\u003cp\u003eThis paper does not report original code.\u003cbr\u003e \u003c/p\u003e\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eWe thank Drs. Wenzhao Wang and Weixin Ke (Institute of Microbiology, Chinese Academy of Sciences) for their advice in metabolome data collection and animal infection. We thank Professor Zhonghua Ma from Zhejiang University for providing the bimolecular fluorescent complementary vectors. We thank Professor Cunwei Cao from Guangxi Medical University for providing clinical isolates of \u003cem\u003eA. fumigatus\u003c/em\u003e. This work was supported by the Strategic Priority Research Program of Chinese Academy of Sciences [grant no. XDB0830000]; the National Natural Science Foundation of China [grant no. 32470046 and 32170066]; the Key Research Program of Frontier Sciences, Chinese Academy of Sciences [grant no. ZDBS-LY-SM016]; the Chinese Academy of Sciences Project for Young Scientists in Basic Research [grant no. YSBR-111].\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eW.-B.Y. conceived the research and supervised the study. Z.S. performed fungal phenotypic analysis, HPLC analysis, transcriptome and metabolome analysis, phylogenetic analysis, subcellular localization, pull-down assay, BiFC assay, structural prediction, molecular docking and murine experiments. H.Z. and Y.L. performed the construction, fermentation, SMs extraction and qRT-PCR experiments of fungal mutants. L.Y. assisted in constructing the infection model. L.W. supervised the murine infection experiments. X.L., C.Z. and K.H.W. analyzed and evaluated the data. N.M.M.S., H.L., and L.C. participated in the discussion of the results. N.P.K., B.R.O., and M.B. revised the manuscript. Z.S. and W.-B.Y. wrote the paper.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eMaterials \u0026amp; Correspondence\u003c/p\u003e\n\u003cp\u003eAll relevant data, including further image and processed data, are available by request from the corresponding author ([email protected]).\u003c/p\u003e\n\u003cp\u003eAdditional information\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary Tables. 1\u0026ndash;7.\u003c/p\u003e\n\u003cp\u003eSupplementary Figs. 1\u0026ndash;9.\u003cbr\u003e \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGangneux, J. P., Hoenigl, M. \u0026amp; Papon, N. 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Here, we uncover a regulatory hub in \u003cem\u003eAspergillus fumigatus\u003c/em\u003e that coordinates virulence through a specialized metabolite network. Using a multi-omics combined chemistry strategy, we identified fumiquinazoline C (FqC) as a keystone metabolite enabling fungal pathogenicity. Central to this process is the RNA-binding protein CsdA, which forms a dynamic nuclear complex with the global regulator LaeB, bridging metabolic remodeling to virulence. Disrupting this complex led to hyperactivation of secondary metabolism, enhancing fungal colonization and virulence. Through deconstructing this hub of CsdA-LaeB-FqC, we pinpointed PptA\u0026mdash;a phosphopantetheinyl transferase essential for secondary metabolite synthesis\u0026mdash;as a linchpin controlling metabolic virulence. Strikingly, FDA-approved drugs (tepotinib, ibrutinib, eltrombopag) targeting PptA suppressed FqC biosynthesis, reduced fungal burden and attenuated lung inflammation in murine models significantly. These findings decode a pathogen\u0026rsquo;s \u0026ldquo;metabolic virulence code\u0026rdquo; and establish a drug-repurposing paradigm to combat antifungal resistance\u003c/p\u003e","manuscriptTitle":"CsdA-LaeB hub governs Aspergillus fumigatus virulence via FqC biosynthesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-12 05:37:51","doi":"10.21203/rs.3.rs-6615529/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"00262acc-d170-4981-8c70-a063597737e0","owner":[],"postedDate":"June 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49921003,"name":"Biological sciences/Microbiology/Fungi"},{"id":49921004,"name":"Biological sciences/Chemical biology"}],"tags":[],"updatedAt":"2025-07-02T13:50:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-12 05:37:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6615529","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6615529","identity":"rs-6615529","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}