Phosphatidylcholine biosynthesis via ChoC is crucial for cellular integrity and virulence in Rhizopus microsporus

ABSTRACT

Rhizopus microsporus is an opportunistic fungal pathogen and a prominent etiological agent of mucormycosis, a life-threatening invasive fungal infection. In this study, we investigated the role of choC, a gene encoding a putative phospholipid methyltransferase involved in phosphatidylcholine (PC) biosynthesis through the Bremer–Greenberg pathway. Deletion of choC using CRISPR-Cas9 led to severe defects in growth and asexual development, which persisted even at 37°C, a temperature relevant to host infection. Supplementation with choline or its precursor PDEA restored growth, confirming the importance of choC in maintaining PC levels. Lipidomic and transcriptomic analyses revealed broad disruptions in PC and phosphatidylethanolamine (PE) levels, glycerol-3-phosphate and fatty acid metabolism, endoplasmic reticulum (ER) stress responses, and vesicle trafficking. Notably, ΔchoC mutants exhibited reduced melanin and chitin content, while ergosterol levels remained unaffected, indicating specific impairment in cell envelope assembly. In a murine infection model, the ΔchoC mutant showed attenuated virulence, linking lipid imbalance to impaired host adaptation. Together, these findings highlight choC as a key regulator of membrane homeostasis and fungal pathogenicity. As the methylation-dependent PC synthesis pathway is barely present in human tissues, choC represents a promising target for selective antifungal therapies.

Introduction

Rhizopus microsporus is a filamentous basal fungus belonging to the subdivision Mucoromycotina and the order Mucorales [1]. It is recognized as a model organism for studying symbiotic interactions between bacteria and fungi [2–4], and has notable industrial applications, such as the production of enzymes and fermented foods [5,6]. However, R. microsporus is also one of the primary opportunistic pathogens responsible for mucormycosis [7], an invasive infection characterized by blood vessel invasion, thrombosis, and tissue necrosis. These features favor fungal dissemination throughout the host [8], resulting in high mortality rates that can reach up to 90% in cases where the infection becomes systemic. Because of this severity, the disease has been colloquially termed “Black Fungus” [9–11]. Species of Rhizopus, along with Mucor and Lichtheimia, account for approximately 90% of mucormycosis cases [11,12]. While this disease mainly affects individuals with diabetes or immunosuppression treated with corticosteroids [13], it can also occur in otherwise healthy individuals following traumatic inoculation [14]. This became particularly evident during the second wave of the COVID-19 pandemic in Asian countries, especially in India, where a significant increase in post-COVID-19 mucormycosis cases was reported [14,15]. Recently, new molecular and genetic strategies based on the CRISPR-Cas9 system have been developed to manipulate the genome of R. microsporus [16], making it the only Mucoral pathogen currently available for genetic modification. These tools enable the study of essential components for mucoralean fungal development, positioning R. microsporus as an ideal model for research and the development of specific and effective antifungal therapies against mucormycosis.

Lipids are a fundamental class of biomolecules essential for life, notable for their structural and functional diversity across biological systems [17]. In 2005, the International Lipid Classification and Nomenclature Committee categorized lipids into eight major categories: fatty acids (FA), glycerolipids (GL), glycerophospholipids (GP), sphingolipids (SL), sterol lipids (ST), prenol lipids (PR), saccharolipids (SL), and polyketides (PK) [18], with FA, GP, SL, and ST being the most common lipid classes in cell membranes [19,20].

Among glycerophospholipids, phosphatidylcholine (PC) is the most abundant component of cell membranes, playing a wide variety of molecular roles (Furse & de Kroon, 2015; Papangelis & Ulven, 2022) [21,22]. There are two main pathways for PC biosynthesis: (i) the Kennedy pathway and (ii) the Bremer-Greenberg pathway or methylation pathway. In mammals (except hepatocytes), nematodes, plants, and malaria parasites, the Kennedy pathway is the predominant route for PC synthesis [23–26]. In contrast, both pathways contribute to PC biosynthesis in fungi and in human hepatocytes, although in the latter, the methylation pathway accounts for only ~30% of PC production [19,27,28]. In Saccharomyces cerevisiae, the Bremer-Greenberg pathway begins with serine, which is decarboxylated to form phosphatidylserine. This intermediate is then converted into PC through three sequential methylation steps, using S-adenosyl-L-methionine as the methyl group donor. The first reaction is catalyzed by Cho2, a class II phospholipid methyltransferase (PLMT), whereas the subsequent two methylation steps are carried out by Opi3, a class I PLMT [12,20,28]. Conversely, the Kennedy pathway is initiated with choline, which is phosphorylated and then conjugated with cytidine triphosphate (CTP) to facilitate its incorporation at the sn-3 position of the glycerol backbone [29]. Additionally, the CDP-DAG route, which originates from glycerol-3-phosphate (G3P), generates precursors shared by both biosynthetic pathways [30,31] (Figure 1(A)).

Figure 1.

(A) Schematic representation of the phosphatidylcholine (PC) biosynthesis pathways in fungi, including the CDP-DAG pathway, the Kennedy pathway, and the Bremer-Greenberg methylation pathway. (b) Gene structure of choC from R. microporus. Red indicates the transcribed exons, while blue corresponds to untranslated regions (UTRs) of the transcript. (C) Comparison of the protein architectures of Opi3 from S. cerevisiae (left) and ChoC from R. microsporus (right). (d) Phylogenetic tree of ChoC orthologs across various fungal species.

Abbreviations: C: Choline, C-P: Phosphocholine, CDP-C: CDP-choline, CDP-DAG: CDP-diacylglycerol, CDP-EA: CDP-ethanolamine, cl: Cardiolipin, DAG: Diacylglycerol, EA: Ethanolamine, EA-P: Phosphoethanolamine, G3P: Glycerol-3-phosphate, LPA: lLysophosphatidic acid, PA: Phosphatidic acid, pc: Phosphatidylcholine, PDEA: Phosphodimethylethanolamine, PE: Phosphatidylethanolamine, PI: Phosphatidylinositol, PMEA: Phosphomonoethylamine, PDP: Phosphatidylglycerophosphate, PGro: Phosphatidylglycerol, PS: Phosphatidylserine.

Several studies have explored the effects of PC disruption in various fungal species. In Aspergillus nidulans and Aspergillus fumigatus, alterations in phosphatidylcholine synthesis affect lipid homeostasis, hyphal development, sporulation, and key cellular processes such as secondary metabolite production and pre-apoptotic induction. However, both fungi show partial recovery after four days [20,32]. In Pestalotiopsis microspora, impairment in PC synthesis affects vegetative growth, sexual structure formation, cell wall integrity, and causes morphological abnormalities. It also reduces stress tolerance and secondary metabolite production, such as taxol, highlighting the critical importance of PC for development, viability, and functionality [12]. In Metarhizium robertsii, disruption of PC synthesis affects lipid homeostasis, disrupts cell polarity, impairs the development of key structures such as the appressorium, and significantly reduces virulence, emphasizing the crucial role of PC in fungal biology, pathogenicity, and infection capability (Gao et al., 2016) [33,34]. In this study, we identified an ortholog of opi3 in R. microsporus, named choC, and demonstrated that this gene is involved in growth, asexual development, melanin and chitin accumulation, lipid biosynthesis, and virulence. These findings position choC as a promising target for the development of novel antifungal strategies against mucormycosis.

Results

Phospholipid methyltransferase-encoding gene choC in R. microsporus

To identify the gene encoding the phospholipid methyltransferase in R. microsporus, the sequence of the single-copy homolog in S. cerevisiae, Opi3 (Uniprot accession No. P05375), was used as a reference to perform a pBLAST search against the R. microsporus genome database available at the Joint Genome Institute (https://mycocosm.jgi.doe.gov/Rhimi59_2/Rhimi59_2.home.html). The identified homolog, designated as ChoC (JGI accession No. 1,882,585), is 930 bp in length and consists of five exons and four introns (Figure 1(B)). The encoded protein, comprising 195 amino acids, corresponds to a class I PLMT, and shares a close protein architecture with Opi3 from S. cerevisiae (Figure 1(C)). At the amino acid level, ChoC shares high identity with other class I PLMTs from both Dikarya and Basal Fungi. For instance, it exhibits 67.88% identity with its homolog in Phycomyces blakesleeanus and 47.15% identity with that of A. fumigatus (Figure 1(D), Figure S1).

To analyze the function of the choC gene in R. microsporus, single mutants ($\mathrm{\Delta }$choC) were generated by CRISPR-Cas9 system. For the single-deletion mutant, fragments containing the pyrF selectable marker were used for genetic transformation and the disruption of the choC gene in the strain UM33 (leuA-, pyrF-). Two transformants selected were homokaryons for the mutant allele because of the difference in band sizes observed in electrophoresis (Figure S2). These knockout mutants were named as follows: UM120.2 and UM120.5 (leuA-, choC:pyrF+).

Δchoc mutants display impaired growth and asexual development

Phenotypic characterization of the $\mathrm{\Delta }$choC mutants revealed significant physiological and morphological differences compared to the wild-type strain UM6 (leuA-, pyrF+). Both mutant strains displayed markedly reduced radial growth on solid medium at both 30°C and 37°C (Figures 2(A) and S3). Importantly, 37°C corresponds to human body temperature, which makes this observation particularly relevant in the context of host-pathogen interactions.

Figure 2.

(A) Colony morphology of wild-type UM6 and $\mathrm{\Delta }$choc mutant strains (UM120.2 and UM120.5) grown on solid minimal medium (MMC, pH 3.2) for 5 days at 30°C (top panels) and 37°C (bottom panels). (b) Colony morphology of wild-type UM6 and $\mathrm{\Delta }$choc strains grown on MMC pH 3.2 supplemented with serine, ethanolamine (ea), phosphomonoethylamine (PMEA), phosphodimethylethanolamine (PDEA), choline or Congo Red, incubated at 30°C for 5 days. Images were captured on day 4. (C) Growth curve of wild-typpe UM6 and $\mathrm{\Delta }$choc strains in liquid MMC over 7 days at 30ºC. The image of the mycelial pellets was taken after 5 days of growth. (d) Quantification of sporulation in wild-type UM6 and $\mathrm{\Delta }$choc strains grown in MMC at 30°C (left) and 37°C (right) for 5 days. Data represent mean ± sd from three biological replicates per condition. padj < 0.001 (***), padj < 0.0001 (****) and ns = not significant.

The addition of PDEA and choline successfully restored colony development in the $\mathrm{\Delta }$choC mutants, whereas other metabolic intermediates such as serine, EA, and PMEA failed to do so (Figure 2(B)). This pattern of rescue indicates a specific metabolic block within the PC biosynthesis pathway, which can only be bypassed by direct supplementation with downstream products. These findings strongly support that the phenotypic defects observed in the mutants are not due just to a general phospholipid imbalance but rather reflect a critical dependency on the de novo synthesis of PC via the methylation pathway. The partial rescue by exogenous choline and PDEA further emphasizes that choC plays a central role in maintaining intracellular PC homeostasis, which is essential for normal growth and cellular function in R. microsporus. Notably, the addition of Congo Red to the solid medium did not further exacerbate the growth defect of the ΔchoC mutants, as their growth remained comparable to that observed under non-supplemented conditions. We propose that this lack of an additive effect may reflects the fact that disruption of choC already compromises membrane integrity through altered PC composition, thereby affecting the same cellular processes targeted by Congo Red (Figure 2(B)). This reinforces the conclusion that choC function is indispensable for maintaining membrane and cell wall integrity.

To quantify the growth impairment, we measured the mycelial dry weight of each strain under liquid culture conditions (Figure 2(C)). We saw a significant reduction in biomass production for both $\mathrm{\Delta }$choC mutants compared to the wild-type UM6, consistent with their impaired radial growth observed in solid media. In addition to reduced biomass, the mutants exhibited altered macroscopic morphology in liquid culture, forming irregular, dispersed mycelial structures rather than the dense, compact pellets of wild-type UM6 (Figure 2(C)). These differences suggest that the loss of choC not only impairs total biomass production but also affects hyphal organization and possibly cell surface properties, contributing to defective aggregation behavior.

In Figure 2(D), sporulation was markedly reduced in both ΔchoC mutants at 30°C and 37°C, indicating that choC is required for efficient asexual development. This defect was particularly striking at 37°C, a temperature relevant to host environments, suggesting that choC-dependent phospholipid metabolism plays a key role in enabling developmental programs under thermal or physiological stress. Together, these results demonstrate that choC is essential for proper phospholipid metabolism, supporting vegetative growth, biomass accumulation, cell wall stability and asexual development, and its disruption leads to a pleiotropic phenotype that consistently manifests across a range of physiologically relevant conditions.

Loss of choC affects melanin production and chitin content without altering ergosterol levels

To further investigate the cellular consequences of impaired PC biosynthesis, we analyzed key components of the fungal cell envelope that could be affected by membrane lipid imbalance Notably, disruption of choC in R. microsporus resulted in a consistent reduction in melanin production in both ΔchoC mutants (Figure 3(A)). This observation suggests that choC activity contributes to normal pigment accumulation, potentially by maintaining membrane organization or supporting vesicle trafficking pathways involved in melanogenesis. Given the essential role of phosphatidylcholine in membrane structure and intracellular transport, its absence may indirectly hinder melanin synthesis or export to the cell surface.

Figure 3.

(A) Melanin production quantified by absorbance at 405 nm. (b) Chitin content assessed using Calcofluor White staining. (C) Ergosterol levels measured in fungal biomass. (D–I) Representative images acquired following Calcofluor staining (excitation at 432 nm): (D) UM6 grown without choline. (E) UM6 grown in the presence of choline. (F) UM120.2 grown without choline. (G) UM120.2 grown in the presence of choline. (H) UM120.5 grown without choline. (I) UM120.5 grown in the presence of choline. All strains were grown on MMC medium supplemented with Congo Red. Data represent mean ± sd from three (melanin and chitin) or four (ergosterol) biological replicates per condition. Statistical significance: padj < 0.01 (**), padj < 0.001 (***), ns = not significant.

Interestingly, quantification of key cell envelope components revealed a selective alteration in the ΔchoC mutants: chitin levels were significantly reduced, while ergosterol content remained comparable to that of the wild-type strain UM6 (Figure 3(B,C)). This suggests that the disruption of choC specifically impairs aspects of cell wall biogenesis without broadly affecting sterol biosynthesis. Chitin is predominantly localized in fungal spores within the sporangium, with higher staining intensity observed in regions adjacent to the peridium, as previously described for Mucorales [35]. Consistent with these quantitative data, calcofluor white staining revealed a clear reduction in chitin-associated fluorescence in the ΔchoC strain under Congo Red stress, providing direct visual evidence of altered chitin distribution. Importantly, supplementation with choline partially restored the calcofluor staining pattern in the ΔchoC mutant, indicating a recovery of chitin deposition under these conditions (Figure 3(D,E,F,G,H,I)), further supporting the notion that the observed phenotype arises from dysregulation of endogenous PC biosynthesis. The reduction in chitin, a major structural polysaccharide in the fungal cell wall, supports the notion that PC biosynthesis via choC is critical for proper cell wall assembly and remodeling, likely through indirect effects on membrane organization, trafficking, or enzymatic regulation of chitin metabolism.

Global gene expression analysis in the $\mathrm{\Delta }$choc mutant

The phenotypic defects observed in the ΔchoC mutant suggest a broader impact on cellular physiology. To investigate the molecular basis of this pleiotropic phenotype, we performed RNA-seq analysis of wild-type UM6 and the ΔchoC mutant at 30ºC and 37ºC in MMC.

A total of 2,834 differentially expressed genes (DEGs) were identified when comparing the ΔchoC mutant and wild-type strain at 30°C (UM12030 vs. UM630), including 1,277 upregulated and 1,557 downregulated genes. At 37°C (UM12037 vs. UM637), a similar number of DEGs was observed (2,838), with 1,516 genes upregulated and 1,322 downregulated in the mutant. Temperature-dependent comparisons within the ΔchoC background (UM12037 vs. UM12030) revealed 2,175 DEGs, with a majority (1,494) being upregulated at 37°C, and 681 downregulated. In contrast, the wild-type strain showed 2,437 DEGs between 30°C and 37°C (UM637 vs. UM630), distributed almost equally between upregulated (1,205) and downregulated (1,232) genes (Figure S4). Among these DEGs, we identified multiple genes involved in both the Kennedy pathway and the Bremer-Greenberg methylation pathway for PC biosynthesis, such as ept1/cpt1, cho2 or ect1; highlighting a transcriptional response directly linked to the disruption of choC. Interestingly, choC expression itself was found to be downregulated in the wild-type strain under elevated temperature conditions (UM637 vs. UM630), suggesting that R. microsporus may naturally modulate choC transcription in response to heat stress (Figure 4(A)).

Figure 4.

(A) Heatmap showing the expression patterns of the genes involved in phospholipid biosynthesis in the wild-type UM6 and $\mathrm{\Delta }$choc mutant (UM120) strains grown at 30°C and 37°C. Data represent triplicate samples per strain and condition. Asterisks (*) indicate statistically significant differential expression, and “x” denotes the specific condition in which significance was observed. Repeated gene names correspond to paralogous genes present in the genome. (B) Venn diagram representing the overlap of DEGs among the four pairwise comparisons. (C) GO enrichment analysis of temperature-dependent genes influenced by the mutant background. (D) GO enrichment analysis of mutant-dependent genes influenced by temperature. (E) GO enrichment analysis of temperature-specific genes. (F) GO enrichment analysis of mutant-specific genes. GO terms are categorized into biological process (BP), cellular component (CC), and molecular function (MF), represented in different colors. GO terms highlighted in yellow indicate those that pass the FDR threshold (FDR < 0.05).

Transcriptomic classification reveals distinct gene expression modules

Given the extensive set of DEGs identified across the previously described comparisons, we next aimed to organize these genes based on their expression dynamics in response to ΔchoC deletion, temperature variation, or their interaction. This approach allowed us to assign each DEG into one of several biologically meaningful categories based on its transcriptional behavior across genotypes and temperatures (Figure 4(B)). These categories include: (i) Mutant-dependent genes influenced by temperature: genes that exhibit transcriptional changes primarily attributable to the ΔchoC deletion, with detectable differences under both temperature conditions. However, the extent, directionality, or statistical significance of these differences varies with temperature. This category may reflect choC-dependent pathways that are differentially modulated by thermal conditions (ii) Temperature-dependent genes influenced by the mutant: genes that are predominantly regulated by temperature shifts in both genotypes. However, the presence of the ΔchoC deletion alters the magnitude or statistical strength of the temperature-dependent response. These genes may represent general thermal stress response pathways that are secondarily affected by the loss of choC (iii) Mutant-specific genes, which are differentially expressed exclusively as a result of ΔchoC deletion, regardless of temperature; (iv) Temperature-specific genes, whose expression is affected solely by changes in temperature, independent of genotype; and (v) Genes with complex interaction (influenced by both temperature and the mutant), which display non-additive or unexpected regulatory patterns reflecting a combined effect of genotype and environmental cues. Additionally, a set of subcategories was defined based on the direction and magnitude of gene regulation, in order to facilitate a more detailed interpretation of transcriptional behavior in specific genotype– temperature contexts (Data Availability, DOI: 10.5281/zenodo.18343892) [75].

To further explore the biological roles of the DEGs, we performed GO term enrichment analysis using the GOseq package (Young et al., 2010). The analysis of temperature-dependent genes influenced by the mutant (Figure 4(C)) revealed enrichment in metabolic and membrane-associated functions, including chitin deacetylase activity (p = 1.06 × 10⁻⁴), oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor (p = 1.16 × 10⁻⁴), and several fatty acid – CoA ligase activities across chain lengths (p = 1.71 × 10⁻³ each). Biological processes such as carbohydrate metabolic process (p = 1.03 × 10⁻⁴), malate metabolic process (p = 7.76 × 10⁻⁴), and lipopolysaccharide biosynthesis (p = 2.75 × 10⁻²) were also enriched. Likewise, mutant-dependent genes influenced by temperature (Figure 4(D)) were associated with acid phosphatase activity (p = 6.60 × 10⁻⁹), oxidoreductase activity (p = 1.20 × 10⁻⁴), monooxygenase activity (p = 2.54 × 10⁻⁴), and phospholipase C activity (p = 2.22 × 10⁻³), alongside processes such as glycerol-3-phosphate metabolism (p = 2.53 × 10⁻³) and catabolism (p = 1.87 × 10⁻²), carbohydrate metabolism (p = 4.40 × 10⁻⁹), transport (p = 6.19 × 10⁻⁴), and membrane/extracellular localization (p = 8.95 × 10⁻⁴ and p = 6.67 × 10⁻³, respectively).

In the case of temperature-specific genes (Figure 4(E)), the analysis identified significant enrichment for cytoskeletal motor activity (p = 4.75 × 10⁻³), ATP-dependent peptidase activity (p = 9.42 × 10⁻³), oxidoreductase activity (p = 1.96 × 10⁻²), and dolichol kinase activity (p = 2.20 × 10⁻²). Enrichment in the myosin complex (p = 4.75 × 10⁻³), pyruvate dehydrogenase complex (p = 2.20 × 10⁻²), and nickel cation transport (p = 2.20 × 10⁻²) suggests engagement of mitochondrial, cytoskeletal, and metal homeostasis systems, reflecting core adaptations to thermal conditions independent of choC status. Finally, mutant-specific genes (Figure 4(F)) were significantly enriched in COPI vesicle coat (p = 5.27 × 10⁻⁴), COPII vesicle coat (p = 1.26 × 10⁻³), and the endoplasmic reticulum (p = 3.52 × 10⁻³), reflecting vesicle-mediated transport pathways essential for lipid trafficking. Further enrichments in protein disulfide isomerase activity (p = 1.03 × 10⁻⁴), structural molecule activity (p = 3.24 × 10⁻⁴), heat shock protein binding (p = 6.96 × 10⁻⁴) indicate that ΔchoC deletion affects protein homeostasis, ER stress, and membrane remodeling. Regulatory functions such as DNA-templated transcription (p = 7.21 × 10⁻³) and electron transfer activity (p = 3.76 × 10⁻²) were also prominent. All significant GO terms are summarized in the Data Availability record (DOI: 10.5281/zenodo.18343892) [75], and detailed GO results for the “Genes with complex interaction” category are provided in Figure S5. In summary, these results demonstrate that deletion of choC disrupts phosphatidylcholine biosynthesis and initiates a coordinated transcriptional response that affects membrane composition, vesicle trafficking, ER function, and cell wall remodeling. These alterations reflect both metabolic stress and adaptive responses, highlighting the central role of PC homeostasis in maintaining cellular integrity and environmental resilience in R. microsporus.

Functional lipid imbalance induced by choC deletion in R. microsporus

To better understand how choC deletion affects lipid metabolism in R. microsporus, we performed a comprehensive lipidomic analysis comparing ΔchoC and wild-type UM6 strains after 24 h of growth in MMC at 30°C and 37°C. Analysis of PC species revealed clear differences both between mutant and wild-type strains and between growth temperatures. Relative to the wild-type UM6 strain, ΔchoC mutants exhibited an increased abundance of shorter-chain PC species (PC 32:1, 32:2, 34:2) together with a concomitant reduction in longer-chain and more unsaturated species (PC 36:4, 38:6) at both temperatures (Figure 5(A)). Within the mutant strain, growth at 37°C partially shifted the PC profile toward longer-chain species compared to 30°C, although the overall PC composition remained distinct from that of the wild-type strain. PE profiles showed a similar pattern of strain- and temperature-dependent differences. Compared to the wild-type strain, ΔchoC mutants displayed reduced levels of several abundant PE species, including PE 36:2, 38:4, and 38:5, while shorter and less unsaturated species such as PE 34:1 and 36:1 were relatively enriched (Figure 5(B)). Notably, comparison between mutant cultures revealed that PE species abundances were partially restored at 37°C relative to 30°C, indicating temperature-dependent remodeling of PE composition specifically in the absence of ChoC.

Figure 5.

(A–D) Heatmaps showing the base peak area of lipid species within the classes PC (A), PE (B), PA (C), and cardiolipin (D) in R. microsporus wild-type UM6 and UM120 ($\mathrm{\Delta }$choc mutant) strains under different temperature conditions. Hierarchical clustering was applied to both lipid species and sample groups. Quantification of PC (E) and PE (F) fragments detected by neutral loss scan of characteristic fragments in wild-type UM6 and $\mathrm{\Delta }$choc mutant strains grown at 30°C and 37°C. Data represent mean ± sd from four biological replicates per condition. padj < 0.01 (**), padj < 0.001 (***), padj < 0.0001 (****), and ns = not significant.

To assess total PC and PE levels, we performed neutral loss scans targeting class-specific fragments. As expected, PC levels in the ΔchoC mutants were drastically reduced at both temperatures, being nearly undetectable at 30°C and only partially recovered at 37°C (Figure 5(E)), consistent with the disruption of methylation-dependent PC biosynthesis. In wild-type strains, PC levels also showed a slight decrease at 37°C, likely reflecting the previously observed downregulation of choC expression at this temperature (Figure 4(A)). PE levels were also significantly reduced in ΔchoC mutants at 30°C, with partial recovery observed at 37°C. In contrast, PE content remained stable across temperatures in wild-type strains, suggesting that temperature-dependent regulation of PE homeostasis is only evident in the absence of ChoC (Figure 5(F)).

Beyond PC and PE, ΔchoC strains also exhibited pronounced changes in phosphatidic acid (PA) and cardiolipin (CL) profiles. Relative to the wild-type UM6 strain, mutants showed increased levels of PA 34:2 and 34:3, whereas longer-chain PA species such as PA 36:2 and 38:5 were more abundant in the wild-type (Figure 5(C)). These differences were consistently observed at both temperatures, with modest temperature-dependent variation within the mutant strain. Similarly, several CL species, including CL 72:2, 72:3, and 74:3, were enriched in ΔchoC mutants, whereas CL 70:5 and 70:4 predominated in the wild-type strain (Figure 5(D)). Comparison between mutant cultures grown at 30°C and 37°C revealed subtle but reproducible shifts in CL species abundance, suggesting that choC deletion impacts cardiolipin remodeling in a temperature-dependent manner.

These results point to broader effects of choC deletion on membrane lipid composition, including potential alterations in mitochondrial function. Hierarchical clustering across lipid classes consistently distinguished mutant from wild-type profiles, highlighting the extensive lipidomic remodeling triggered by choC disruption.

Disruption of phosphatidylcholine biosynthesis affects virulence in R. microsporus

To evaluate the roles of choC in the virulence of R. microsporus, we conducted a survival assay in a murine infection model using immunocompetent mice. Four groups of ten mice each were infected with the parental strain ATCC 11,559, wild-type UM6, the avirulent recipient strain UM33, and the ΔchoC mutant. As shown in Figure 6, UM33 caused no mortality, with all animals surviving up to 10 days post-infection (DPI), confirming its nonpathogenic phenotype. In contrast, both ATCC 11,559 and UM6 were highly virulent, resulting in 100% mortality by day 6 DPI. Infection with the UM120 strain revealed a clearly attenuated virulence phenotype compared to those infected with UM6. Kaplan–Meier survival curves were compared using the log-rank (Mantel–Cox) test, revealing significant differences between UM120 and UM6 (p = 0.0063) as well as between UM120 and ATCC 11,559 (p = 0.0037), whereas no significant difference was observed between ATCC 11,559 and UM6 (p = 0.6442). Although all mice eventually succumbed to infection, the onset of mortality was delayed, and overall survival times were significantly prolonged compared to those infected with UM6 or ATCC 11,559.

Figure 6.

Survival curve of mice infected with R. microsporus over 10 days. Parental strain 11,559 served as the positive virulence control, UM33 as the negative control, UM6 as the wild-type background, and UM120 as the $\mathrm{\Delta }$choc mutant. Significant differences were observed between 11,559 and UM33 (p < 0.0001), 11,559 and UM120 (p = 0.0037), UM33 and UM6 (p < 0.0001), and UM6 and UM120 (p = 0.0063). No significant difference was detected between 11,559 and UM6 (p = 0.6442).

This attenuation in virulence indicates that disruption of choC compromises the pathogenic potential of R. microsporus, likely by altering phospholipid metabolism and thereby affecting essential cellular processes required for infection. The phenotype observed in the ΔchoC mutant underscores the critical role of phospholipid homeostasis in virulence and highlights ChoC as a potential target for the development of novel therapeutic strategies against mucormycosis.

Methodology

Fungal strains and growth conditions

All fungal strains used and generated in this work were derived from R. microsporus ATCC 11,559 (Table S1). When needed, the media were supplemented with uridine (200 mg/L) or leucine (20 mg/L). Transformants using the pyrF marker were grown in Yeast Nitrogen Base (YNB; 1.5 g/L ammonium sulfate, 1.5 g/L glutamic acid, 0.5 g/L yeast nitrogen base without amino acids and ammonium sulfate, 10 g/L glucose, 15 g/L agar) medium, pH 3.2, supplemented with 1 mL of thiamine (1 mg/mL) and niacin (1 mg/mL). During transformation, electroporated protoplasts were resuspended in ice-cold Yeast Peptone Glucose (YPG; 3 g/L yeast extract, 10 g/L peptone, 20 g/L glucose, 15 g/L agar) medium supplemented with 0.5 M Sorbitol, pH 4.5, for 90 min and then centrifuged at 91 × g and resuspended in YNB supplemented with 0.5 M Sorbitol. Minimal Media with Casamino acids (MMC; 10 g/L Casamino acid, 0.5 g/L yeast nitrogen base without amino acids and ammonium sulfate, 20 g/L glucose, 15 g/L agar) pH 3.2, supplemented with niacin (1 mg/mL) and thiamine (1 mg/mL) was used as the basic medium, and supplements were added according to our experimental design. To explore the function of choC in the methylation pathway, 1000 spores were grown in MMC with 50 mg/L L-serine, 40 mg/L ethanolamine, 50 mg/L N-methylaminoethanol, 60 mg/L N, N-dimethylaminoethanol, 50 mg/L choline chloride, and 2.5 mg/mL Congo red. For liquid-submerged culture, 1000 spores were inoculated into 10 mL liquid medium. To quantify sporulation, 1 cm² of colony grown on MMC (from 1000 spores) was suspended in 5 mL of distilled water, and spores were counted using a Thoma chamber. For the quantification of mycelial dry weight, mycelia were harvested from the solid medium at defined time points during cultivation. Samples were freeze-dried using a lyophilizer until a constant weight was obtained. All cultures were incubated at 30°C under standard indoor light conditions. Liquid cultures were maintained at 250 rpm with continuous shaking. Protein 3D structure predictions were generated using AlphaFold v2.0 [36] via the web-based interface at https://alphafoldserver.com/.

Molecular phylogenetic analysis

Protein sequences were aligned using MAFFT v7 with the L-INS-i algorithm as implemented in the NGPhylogeny platform, under default gap opening and extension penalties and using the BLOSUM62 substitution matrix (Lemoine et al., 2019; Dereeper et al., 2008) [37–39]. The resulting alignment was subsequently curated with BMGE, also through NGPhylogeny, to remove poorly aligned and highly variable regions, using a sliding window size of 3, a maximum entropy threshold of 0.5, a gap rate cutoff of 0.3, and a minimum block size of 5 [38–40]. Phylogenetic reconstruction was performed using the IQ-TREE web server under a maximum likelihood framework, applying the LG substitution model with gamma-distributed rate heterogeneity and a proportion of invariant sites (LG+G+I) [41,42]. Branch support was assessed using 1,000 ultrafast bootstrap replicates and 1,000 SH-aLRT tests [43]. The resulting tree was midpoint-rooted and visualized and annotated using iTOL [44].

Genomic DNA extraction

Genomic DNA was extracted from approximately 500 mg of R. microsporus mycelium ground in liquid nitrogen. The resulting powder was mixed with 1 mL of TE buffer (200 mM Tris-HCl, 100 mM EDTA, 1% SDS), 1 mg of RNase A, and 1 mL of phenol (pH 8.0), followed by gentle mixing and centrifugation (5 min at 4,000 rpm). The aqueous phase was recovered and washed twice with phenol:chloroform:isoamyl alcohol (25:24:1) and once with chloroform:isoamyl alcohol. DNA was precipitated with 5 M ammonium acetate and cold absolute ethanol (−70°C), collected as a visible spool, washed with 70% ethanol, air-dried at room temperature, and resuspended in 100–150 µL of nuclease-free water.

Mutant strain generation and validation

R. microsporus mutant strains were generated based on previously established protocols, with minor modifications (Figure 1). Cell wall digestion was performed using 20 U of Zymolyase Ultra (Zymo Research, Irvine, CA, USA) and 0.0008 U of chitosanase (Merck KGaA, Darmstadt, Germany) per 1 × 10⁷ spores, incubated for 1 hour and 45 minutes at 30°C under gentle agitation. For the disruption of the choC gene, a DNA repair template carrying the pyrF gene flanked by 38-nucleotide homology arms was used to replace the target locus. Gene editing was facilitated by a CRISPR/Cas9-mediated double-strand break (DSB) in the uracil- and leucine-auxotrophic strain UM33 (Figure S2). The CRISPR/Cas9 system was directed using Alt-R™ CRISPR crRNAs (IDT, Integrated DNA Technologies, Coralville, IA, EE.UU) specifically designed to target choC. crRNAs were annealed with Alt-R™ tracRNA (IDT) to form guide RNAs (gRNAs), which were assembled in vitro with Alt-R™ S.p. Cas9 nuclease (IDT) to generate ribonucleoprotein (RNP) complexes. Gene disruptions were validated by PCR using one primer annealing within the marker gene and another upstream of the recombination region. Karyotype status (homo- or heterokaryotic) was assessed by PCR using primers flanking the disrupted locus, yielding products from both wild-type and mutant nuclei when present. All primers used for construct generation and PCR validation are listed in Table S2. Genomic DNA was extracted as previously described, and PCR reactions were performed using Herculase II Fusion DNA polymerase (Agilent Technologies, Santa Clara, CA, EE.UU), with annealing temperatures optimized according to each primer pair following the manufacturer’s recommendations.

Ergosterol and melanin quantification

To obtain fungal biomass for ergosterol and melanin quantification, 50 μL of spore suspension at a concentration of 5 × 10⁵ spores/mL was inoculated into 250 mL of liquid MMC medium. Cultures were incubated at 30ºC with shaking at 125 rpm for 7 days. The resulting biomass was subsequently filtered through monodur filters, washed with sterile distilled water, dried with filter paper, and lyophilized to eliminate all moisture. Finally, the dry biomass was then pulverized with the help of a sterile mortar [45]. The ergosterol quantification was carried out according to the method established by Arthington-Skaggs et al. [46]. Briefly, 60 mg of the powdered biomass were placed into eppendorf tubes and transferred to 10 mL glass tubes containing 6 mL of 25% KOH/EtOH solution. The samples were vortexed and incubated at 85°C in a water bath for 1 h, shaking every 15 minutes. Then, they were cooled to room temperature for 10 minutes, and 2 mL of sterile distilled water and 6 mL of n-heptane were added. The samples were homogenized for 3 minutes to allow the interaction between ergosterol and n-heptane, and the upper layer generated by said compounds was transferred to glass tubes. Finally, quantification was carried out by measuring the absorbance at 281.5 nm and 230 nm of the samples and a blank (n-heptane) using a NanoDrop spectrophotometer and applying the formulas detailed in the referenced methodology [46]. Melanin extraction was conducted using an alkaline method described by Gupta & Chattoo [47]. A total of 20 mg of lyophilized mycelia were weighed in eppendorf tubes, and 0,5 mL of 1 N NaOH solution with 10% DMSO was added to each tube. The samples were incubated in incubator tubes with agitation for 1 h, and then the samples were centrifuged at 12.000 g for 10 minutes. The supernatant was transferred to a new tube, and the absorbance was measured at 405 nm using a NanoDrop spectrophotometer.

Chitin quantification by fluorescence

Chitin content was quantified following the protocols described by Szebenyi et al. and Maybruck et al. [48,49], with modifications. Fresh R. microsporus spores were collected, washed twice in PBS buffer, and centrifugated at 3500 g for 10 minutes, and the cell concentration was diluted to 1 × 10⁷ spores/mL in PBS. Calcofluor White (CFW) was added for a final concentration of 5 μg/ml of CFW solution. Samples were incubated at room temperature for 15 minutes in the dark. After staining, samples were washed and resuspended in PBS. Cells were deposited onto a slide and examined by epifluorescence using filter sets for DAPI (excitation 350 nm; emission 432 nm). Images from three biological replicates were captured and fluorescence intensity measured using the software ImageJ [50].

Rnaseq analysis

Total RNA from three biological replicates per condition was extracted using the NZY Total RNA Isolation kit (MB13402, NZYTech, Lisbon, Portugal), from 1 × 10⁶ spores grown in MMC 3.2 medium for 24 hours, following the manufacturer’s instructions. RNA concentration and integrity were assessed using a Qubit fluorometer (Thermo Scientific) and agarose gel electrophoresis, with the 28S/18S rRNA ratio used as a quality indicator (Table S3). Filtered raw reads were aligned to the reference genome of R. microsporus ATCC 11,559 (available at the JGI MycoCosm Portal: https://mycocosm.jgi.doe.gov/Rhimi59_2/) [51] using HISAT2 (v2.2.0) [52]. Gene-level raw counts were generated with featureCounts (v2.0.3) [53]. Differential gene expression analysis was carried out using DESeq2 (v1.30.0) [54], with statistical significance defined as p < 0.05 and an absolute log fold change (|logFC|) >1. Genes involved in PC synthesis were selected for heatmap analysis, and the complete list is available in Table S5. Gene Ontology (GO) enrichment analysis was performed using the GOseq R package [55], which accounts for gene length bias. GO terms with adjusted p-values < 0.05 were considered significantly enriched.

Lipid extraction

Lipid extraction was performed from approximately 100 mg of frozen mycelium per sample, using four biological replicates per condition (Table S4). All steps were conducted under low-light conditions, on ice, and within a laminar flow hood to prevent lipid oxidation. Samples were resuspended in 300 µL of 1× PBS, followed by the addition of 7 mL chloroform:methanol (2:1, v/v) and vortexed thoroughly. After 1 hour of incubation on ice and protected from light, 2 mL of 0.88% KCl were added, vortexed briefly, and centrifuged at 390 × g for 5 minutes at 4°C. The aqueous phase was carefully removed, and the remaining organic phase was filtered to eliminate protein contaminants. Lipids were then dried under nitrogen at 37°C, weighed, and resuspended in 400 µL of chloroform:methanol (2:1, v/v) containing 0.01% BHT. Final extracts were filtered through 0.22 µm syringe filters and stored at −20°C in amber vials.

High performance liquid chromatography-mass spectrometry (HPLC-MS)

The separation and analysis of samples were performed with a HPLC/MS system consisting of an Agilent 1290 Infinity II Series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with an Automated Multisampler module and a High Speed BinaryPump, and connected to an Agilent 6550 Q-TOF Mass Spectrometer (Agilent Technologies, Santa Clara, CA, USA) using an Agilent Jet Stream Dual electrospray (AJS-Dual ESI) interface. Experimental parameters for HPLC and Q-TOF were set in MassHunter Workstation Data Acquisition software (Agilent Technologies, B.08.00). Samples (20 µl) were thermostatted at 5 ºC and injected onto an Agilent Zorbax Eclipse Plus (2.1 ×100 mm, 1.8 um) HPLC column, equilibrated at 55 ºC. Compounds were eluted at a flow rate of 0.3 ml/min with an isocratic gradient consisting of 60% (A) MilliQ water/acetonitrile (40:60, v:v) and 40% (B) acetonitrile/isopropanol (10:90, v:v) containing 0.1% formic acid and 5 mM acetamide as mobile phases. The mass spectrometer was operated in both positive and negative mode. The nebulizer gas pressure was set to 30 psi, whereas the drying gas flow was set to 16 l/min at a temperature of 130 ºC, and the sheath gas flow was set to 11 l/min at a temperature of 300 ºC. In positive mode analysis, the capillary spray, nozzle, fragmentor and octopole 1 RF Vpp voltages were 4000 V, 500 V, 360 V and 750 V respectively. In the negative mode analysis, voltages were 4000 V, 100 V, 360 V and 750 V respectively. Centroid data in the 50–1200 m/z range were acquired for MS scans in 2 GHz Extended Dynamic Range High Resolution mode with 4 spectra/s, 250 ms/spectrum and 1990 transients/spectrum. Reference masses at 121.0509 and 922.0098 m/z (positive mode) and 112.9856 and 1033.9881 m/z (negative mode) were used for mass correction during the analysis. Data analyses were performed with MassHunter Qualitative Analysis Navigator software (Agilent Technologies, B.08.00).

Ethical statement

All animal experiments were conducted in compliance with the European Union Council Guidelines (Directive 2010/63/EU) and the Spanish legislation (RD 53/2013). Protocols were reviewed, approved, and supervised by the Animal Welfare and Ethics Committee of the University of Murcia and the Council of Water, Agriculture, Farming, Fishing and Environment of Murcia (Consejería de Agua, Agricultura, Ganadería, Pesca y Medio Ambiente de la CARM), Spain (authorization number REGA ES300305440012-A13180108). The project was authorized to Dr. Victoriano Garre as the responsible investigator under identification code A13180108.

Mice infection experiments

Male Swiss mice (30 g, one month old; Animal Facilities Services, University of Murcia, Spain) were used for virulence assays. Animals were housed in four groups (ATCC11559, UM6, UM33, and UM120) of ten per cage with food and water ad libitum, under specific-pathogen-free conditions. R. microsporus strain UM33 was used as an avirulent strain for the negative control. Mice were challenged intravenously by retroorbital injection with 1 × 10⁶ sporangiospores in 50 μL PBS per animal. Prior to challenge, animals were anesthetized with inhaled isoflurane using an induction chamber and monitored until full recovery from anesthesia. Animals were examined twice daily for 10 days (17–27 March 2024). Survival rates were plotted in a Kaplan–Meier curve, and differences were considered statistically significant at p < 0.05 using a Mantel–Cox test. At the end of the study, or when humane endpoints were reached, mice were euthanized by CO₂ inhalation in accordance with the AVMA Guidelines for the Euthanasia of Animals (2020). Humane endpoints included severe clinical signs or a loss of more than 25% of the initial body weight; affected animals were euthanized and recorded as having died on the following day. This study was designed, conducted, and reported in accordance with the ARRIVE 2.0 guidelines

Data analysis

All experiments were randomized, and the investigators performing the quantifications were blinded to the experimental conditions. Statistical analyses were conducted using GraphPad Prism version 8.0.1 (GraphPad Software, CA, USA). Data were analyzed by two-way analysis of variance (two-way ANOVA) followed by Turkey’s multiple comparisons test to evaluate the effects of two independent variables and their interaction, or one-way ANOVE followed by Turkey’s multiple comparisons test to evaluate the effects of just one independent variable. A p value < 0.05 was considered statistically significant. Data are presented as mean ± standard deviation (SD) unless otherwise specified.

Discussion

In this study, we characterized the role of choC, which encodes a putative phospholipid methyltransferase, in the filamentous fungus R. microsporus. Deletion of choC resulted in pronounced defects in vegetative growth and sporulation in both solid and liquid media (Figure 2(A,C,D)), underscoring the importance of phosphatidylcholine (PC) biosynthesis for normal fungal development. Supplementation with choline or its precursor phosphodimethylethanolamine (PDEA) restored normal growth, confirming that the observed phenotypes stem from impaired PC production. Similar growth defects associated with disruptions in phospholipid methylation have been reported in other fungi, although the extent of phenotypic recovery varies across species [20,56]. In A. fumigatus and A. nidulans, the phenotype is partially restored after four days of growth, suggesting some compensation by the Kennedy pathway or CDP-DAG pathway [20]. In contrast, no recovery is seen in R. microsporus, P. microsporus or M. robertsii, indicating that choC-dependent PC biosynthesis is essential in these species [12,34]. These findings reinforce our conclusion that the contribution of the Bremer–Greenberg pathway, and the functional relevance of choC, varies across the fungal kingdom. Interestingly, this phenotype is not restricted to a specific temperature, as previously characterized, but is also evident at 37°C, a temperature highly relevant in the context of host–pathogen interactions. The inability of the mutant to grow properly at host temperature suggests that choC-dependent PC biosynthesis may be critical not only for environmental adaptation but also for fungal pathogenicity, asexual development, and cellular function in R. microsporus.

Surprisingly, no significant differences were observed in the R. microsporus ΔchoC mutant when grown in the presence of Congo Red compared to the control condition without the dye (Figure 2(B)). This contrasts with A. fumigatus, where choC mutants display clear hypersensitivity to Congo Red [20]. In R. microsporus, the mutant already exhibits marked growth defects under normal conditions, suggesting that the cell wall is sufficiently compromised such that additional stress from Congo Red has little or no additive effect. Supporting this, quantification of cell wall components revealed a significant reduction in chitin content in ΔchoC strains, whereas ergosterol levels remained unchanged relative to the wild-type UM6 (Figure 3(B,C)). This selective depletion of chitin indicates that choC disruption affects specific aspects of wall biosynthesis, likely through indirect effects on membrane organization, vesicle trafficking, or lipid-mediated regulation of cell wall enzymes. Consistent with the quantitative measurements, calcofluor white staining revealed a clear reduction in chitin-associated fluorescence in the ΔchoC mutant under Congo Red stress (Figure 3(D,E,F,G,H,I)), providing direct visual evidence of altered chitin distribution. Importantly, supplementation with choline restored the calcofluor staining pattern, supporting the idea that impaired endogenous phosphatidylcholine biosynthesis underlies the observed cell wall defects. Similar findings have been reported in Candida albicans, where disruption of phospholipid homeostasis leads to defective chitin deposition without altering ergosterol levels [57]. As PC does not directly form part of the cell wall, its role is most likely mediated through its influence on intracellular signaling and membrane dynamics [19,58]. Consistent with this, transcriptomic analysis in the ΔchoC mutant revealed enrichment of a GO term related to chitin deacetylase activity (Figure 4(C)), suggesting ongoing remodeling of the cell wall structure under conditions of lipid imbalance. These results reinforce the role of ChoC in maintaining cell wall integrity and highlight species-specific differences in compensatory responses to envelope stress.

Transcriptomic analysis of the ΔchoC mutant reveals a coordinated disruption of membrane-associated systems and core metabolic pathways, underscoring the central role of PC biosynthesis in maintaining intracellular homeostasis in R. microsporus. Gene enrichment patterns highlight the impact of choC deletion on multiple fronts, beginning with vesicle trafficking between the ER and Golgi apparatus, which are the primary sites of PC synthesis [19]. Specifically, the differential expression of genes involved in COPI and COPII vesicle coat formation and intra-Golgi vesicle-mediated transport suggests that PC is essential for vesicle budding, cargo sorting, and membrane curvature, all of which are critical for maintaining secretory pathway integrity [59,60]. In addition, functional enrichment in protein disulfide isomerase activity, chaperone binding, and structural molecule activity indicates potential ER stress [61,62], also found in A. fumigatus [20]. Importantly, choC deletion also impacted G3P metabolism, particularly among mutant-dependent genes influenced by temperature. G3P serves as a key metabolic hub that connects glycolysis, lipid biosynthesis (via the Kennedy and Bremer–Greenberg pathways), and redox regulation [19,63]. Altered regulation of G3P-related genes suggests a metabolic shift to compensate for disrupted PC biosynthesis, possibly through the redirection of carbon flux or alternative lipid remodeling strategies [63,64]. This aligns with the enrichment of multiple fatty acid–CoA ligase genes of varying chain-length specificity, indicating broad reprogramming of fatty acid activation and routing [65]. These results position ChoC as a key integrator of phospholipid synthesis with intracellular organization, suggesting that PC is not only a structural lipid, but also a regulator of organelle function and cellular adaptability under environmental stress.

Transcriptomic and lipidomic analyses together revealed that ChoC plays a central and non-redundant role in PC biosynthesis in R. microsporus. In ΔchoC mutants, we observed transcriptional upregulation of cho2, pct1, and ept1/cpt1 paralogs (Figure 3(A)), genes associated with the Kennedy pathway and early methylation steps, suggesting that the cell attempts to redirect lipid metabolism toward PC synthesis via alternative routes. However, in the absence of ChoC, which catalyzes the final methylation step, this compensatory response is ineffective. As a result, PE is consumed, but PC is not produced efficiently, leading to a reduction in both PE and PC levels, as confirmed by neutral loss scan data (Figure 4(E,F)). Interestingly, in the wild-type strain, cho2 expression remains unchanged between 30°C and 37°C, and PE levels are similarly stable across temperatures. In contrast, choC expression is specifically downregulated at 37°C, a pattern that aligns with a mild decrease in PC content, suggesting that R. microsporus actively reduces choC expression under host-like conditions, possibly as a strategy to increase membrane fluidity during infection [66]. However, in the ΔchoC background, this regulation is completely lost, resulting in persistent disruption of lipid balance and inability to adapt membrane composition to temperature shifts. Importantly, our data demonstrated that, although the Kennedy pathway is transcriptionally active, it is insufficient to compensate for the loss of choC. In line with this, total PC levels dropped more than 21-fold at 30°C and 8-fold at 37°C in the ΔchoC mutant compared to wild type (Figure 5(E,F)). These findings highlight that, in R. microsporus, the Bremer–Greenberg pathway is not only essential but also the dominant route for PC synthesis under the tested conditions, acting in collaboration with, but functionally outweighing, the Kennedy pathway.

The attenuated virulence observed in the ΔchoC mutant supports a functional link between phospholipid metabolism and pathogenicity in R. microsporus. Although all infected mice ultimately succumbed, the delayed mortality indicates that loss of ChoC compromises key physiological processes required for efficient host invasion and progression of infection. Similar connections between lipid metabolic pathways and fungal virulence have been described in multiple pathogenic fungi, including C. albicans, A. fumigatus, and M. robertsii, where mutations affecting phospholipid biosynthesis impair growth, cell wall integrity, and pathogenicity [20,34,55].

One contributing factor is the marked reduction in melanin production observed in ΔchoC strains. Melanin is a well-established virulence factor that protects fungi from oxidative stress, phagocytosis, and antimicrobial compounds [67]. Similar impairments have been reported in melanin-deficient mutants of A. fumigatus and R. delemar, which show reduced survival in immune cells and decreased virulence [68,69]. Given that choC affects membrane lipid composition, these defects may arise from altered vesicular trafficking of melanogenic enzymes or intermediates. This notion is supported by our transcriptomic data, which revealed disruptions in genes involved in vesicle formation and transport, reinforcing the idea that phosphatidylcholine is crucial for proper trafficking and secretion processes.

Targeting lipid metabolism has emerged as a promising antifungal strategy, particularly for pathogens that exhibit intrinsic resistance to current drug classes. Recent reviews highlight fungal lipid biosynthesis pathways as attractive targets for antifungal drug development and for potentiating existing therapies [70,71]. Additionally, a growing body of work underscores the importance of understanding fungal lipid biology for the discovery of novel antifungal targets [72]. In this context, inhibition of ChoC may represent a strategy to weaken fungal fitness and pathogenic potential, either as a standalone approach or in combination with current antifungals, rather than acting as a fungicidal target on its own. Finally, given the substantial challenges associated with mucormycosis research, including limitations in histopathological resolution and host–pathogen modeling [73], the identification of molecular targets that simultaneously affect multiple virulence-associated processes is of particular interest. While further studies will be required to assess druggability, specificity, and in vivo efficacy, our findings position ChoC as a promising candidate for future antifungal development efforts aimed at expanding the currently limited therapeutic arsenal against mucormycosis.

In summary, choC is a central regulator of PC biosynthesis in R. microsporus, and its disruption leads to widespread metabolic and cellular defects. These include reduced PC and PE levels, altered glycerol-3-phosphate and fatty acid metabolism, ER stress, impaired vesicle trafficking, and compromised cell wall remodeling. The mutant also showed reduced melanin and chitin content, suggesting defects in membrane-dependent secretion and envelope assembly. Collectively, these alterations impair growth, sporulation, and virulence under host-like conditions. As R. microsporus is one of the main causative agents of mucormycosis, the functional conservation of choC in filamentous fungi and the limited role of the phospholipid methylation pathway in mammals (restricted mainly to hepatocytes and contributing only ~30% of total PC synthesis) [19], ChoC emerges as a selective and attractive antifungal drug target for combating mucormycosis.

Funding Statement

This work was supported by the Fundación Séneca – Agencia de Ciencia y Tecnología de la Región de Murcia [21969/PI/22]; and by project [PID2021-124674NB- I00], funded by MICIU/AEI/10.13039/501100011033/ and by FEDER “Una manera de hacer Europa”..

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All data supporting this study are available in the Zenodo repository. The phenotypic and cell-wall/membrane measurements (growth, Congo Red, sporulation, 30°C dry weight; melanin, chitin, ergosterol; Excel workbooks) are available at DOI: https://zenodo.org/records/18336917 [74]. The raw RNA-seq data, processed analysis result and GO-term enrichment outputs generated in this study have been deposited in Zenodo under accession DOI: 10.5281/zenodo.18343892 [75]. Quantitative lipidomic reports (LC–QTOF-MS) are available in Zenodo under accession DOI: https://doi.org/10.5281/zenodo.17072371 [76]. The dataset from murine infection experiments has been deposited in Zenodo under the accession DOI 10.5281/zenodo.17101009 [77].

Supplemental data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2026.2654119