α-1,3-Glucan-Driven Remodeling of the Conidial Cell Wall in an Aspergillus fumigatus Vaccine Strain Alters Innate Immune Recognition.

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Learn more: PMC Disclaimer | PMC Copyright Notice J Am Chem Soc . 2026 Mar 26;148(13):14320–14332. doi: 10.1021/jacs.6c00915 Search in PMC Search in PubMed View in NLM Catalog Add to search α‑1,3-Glucan-Driven Remodeling of the Conidial Cell Wall in an Aspergillus fumigatus Vaccine Strain Alters Innate Immune Recognition Kalpana Singh Kalpana Singh † Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, United States Find articles by Kalpana Singh † , Ankur Ankur Ankur Ankur † Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, United States Find articles by Ankur Ankur † , Jayasubba Reddy Yarava Jayasubba Reddy Yarava † Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, United States Find articles by Jayasubba Reddy Yarava † , Caroline Mota Fernandes Caroline Mota Fernandes ‡ Department of Microbiology and Immunology, Stony Brook University, Stony Brook, New York 11794, United States Find articles by Caroline Mota Fernandes ‡ , Gianluca Vascelli Gianluca Vascelli § Department of Medicine and Surgery, University of Perugia, Perugia 06123, Italy Find articles by Gianluca Vascelli § , Alessia Sulla Alessia Sulla § Department of Medicine and Surgery, University of Perugia, Perugia 06123, Italy Find articles by Alessia Sulla § , Teresa Zelante Teresa Zelante § Department of Medicine and Surgery, University of Perugia, Perugia 06123, Italy Find articles by Teresa Zelante § , Maurizio Del Poeta Maurizio Del Poeta ‡ Department of Microbiology and Immunology, Stony Brook University, Stony Brook, New York 11794, United States ∥ Division of Infectious Diseases, Stony Brook University, Stony Brook, New York 11794, United States ⊥ Veterans Affairs Medical Center, Northport, New York 11768, United States Find articles by Maurizio Del Poeta ‡, ∥, ⊥, * , Tuo Wang Tuo Wang † Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, United States Find articles by Tuo Wang †, * Author information Article notes Copyright and License information † Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, United States ‡ Department of Microbiology and Immunology, Stony Brook University, Stony Brook, New York 11794, United States § Department of Medicine and Surgery, University of Perugia, Perugia 06123, Italy ∥ Division of Infectious Diseases, Stony Brook University, Stony Brook, New York 11794, United States ⊥ Veterans Affairs Medical Center, Northport, New York 11768, United States * Email: [email protected] . * Email: [email protected] . Received 2026 Jan 19; Accepted 2026 Mar 23; Revised 2026 Mar 20; Collection date 2026 Apr 8. © 2026 The Authors. Published by American Chemical Society This article is licensed under CC-BY 4.0 PMC Copyright notice PMCID: PMC13067271  PMID: 41883285 Previous version available: This article is based on a previously available preprint posted on bioRxiv on January 2, 2026: " α-1,3-Glucan-Driven Remodeling of the Conidial Cell Wall in an Aspergillus fumigatus Vaccine Strain Alters Innate Immune Recognition ". Abstract Aspergillus fumigatus is a major cause of invasive aspergillosis in immunocompromised patients, where current antifungal therapies are limited by toxicity, drug resistance, and lack of durable protection, and no vaccines are available. A mutant lacking the sterylglucosidase-encoding gene ( sglA ) has emerged as a candidate that induces protective immune responses, but the structural basis for this phenotype remains unclear. Here, we use cellular solid-state NMR spectroscopy to compare the organization of the conidial cell wall in Δ sglA and its wild-type counterpart. The Δ sglA conidial cell wall displays extensive remodeling, including increased α-1,3-glucan content and structural polymorphism, strengthened interactions with β-glucans, reduced hydration, and restricted molecular motion, together consolidating a more rigid scaffold with limited β-glucan accessibility. These structural changes are associated with altered neutrophil responses and a shift in innate immune signaling. This work links cell–wall reorganization to altered immune recognition in this vaccine candidate, with implications for future immunotherapeutic strategies. Introduction Aspergillus fumigatus is the major etiological agent of invasive aspergillosis, a life-threatening fungal infection that affects approximately 200,000 people worldwide each year and is associated with a mortality rate of 30–90%. , The disease primarily affects immunocompromised individuals, including patients with AIDS, organ or stem-cell transplants, and those receiving immunosuppressive therapies. , Infection is initiated by inhalation of airborne conidia, which reach the lung parenchyma and germinate into invasive hyphae. − In immunocompetent hosts, the innate immune response is generally sufficient to clear the fungus; however, in individuals with impaired immunity, the infection progresses to invasive disease. − Azole antifungal drugs, which inhibit ergosterol biosynthesis, remain the first-line therapy for invasive aspergillosis. − Their clinical effectiveness, however, has been increasingly compromised by the emergence and global spread of azole-resistant A. fumigatus strains. − Echinocandins, a newer class of antifungals that inhibit β-1,3-glucan synthesis in the fungal cell wall, are also used in clinical practice. − Although effective in limiting fungal growth, echinocandins are largely fungistatic rather than fungicidal against Aspergillus species, and thus are primarily employed as second-line agents in invasive aspergillosis. − Compounding these therapeutic limitations, there is currently no antifungal vaccine licensed for the clinical prevention or treatment of invasive aspergillosis. , This challenge is further exacerbated by the fact that invasive aspergillosis primarily affects immunosuppressed patients, whose reduced immune-cell function compromises both natural host defenses and the effectiveness of potential vaccination strategies. , In response to this unmet need, vaccination-based approaches have gained increasing attention. Several studies have demonstrated that vaccination with viable Aspergillus conidia can elicit protective immunity, with up to 70% of immunocompromised mice surviving lethal challenge following immunization. , Building on this concept, a gene-deletion strategy targeting sterylglucosidase (SGL1) in Cryptococcus neoformans generated a Δ sgl1 mutant that accumulates sterylglucosides and confers complete protection against lethal fungal infections across multiple immunosuppression models. , A similar phenotype was observed in A. fumigatus , where the Δ sglA mutant exhibited attenuated virulence during primary infection and was fully cleared from the lungs of immunocompromised mice. These advances suggest that fungal sterylglucosides, together with associated cell–wall alterations, may represent a promising immunomodulatory platform for vaccine development in vulnerable hosts. However, the mechanisms underlying the avirulence of the vaccine-candidate strain, the A. fumigatus Δ sglA mutant, remain poorly understood, particularly with respect to how cell–wall remodeling and sterylglucoside accumulation contribute to its immunoprotective response. In this study, we employ 13 C and 1 H-detected solid-state NMR to uncover the unique structural features of the cell wall in intact Δ sglA conidia. This nondestructive spectroscopic approach enables the resolution of the structure, dynamics, and physical interactions of polysaccharides in living cells. − This capability has recently been leveraged to elucidate adaptive cell–wall remodeling across diverse fungal pathogensincluding Candida species, Cryptococcus species, and filamentous fungi such as Aspergillus and Mucor speciesin systems of major clinical and biological relevance. − High-resolution analyses of Δ sglA conidia reveal a distinct molecular architecture characterized by an unusually rigid and dehydrated cell wall, with markedly increased α-1,3-glucan incorporated into the rigid structural scaffold. We further correlate this α-1,3-glucan-mediated masking of β-glucans with the loss of Dectin-1 signaling in neutrophils, thereby bridging the gap between cell–wall remodeling and immune responses that underlie the functional mechanism of this vaccine candidate, and providing structural insights that may inform the future development of antifungal vaccines. Result Structural Features of Polysaccharides in the Organization of A. fumigatus Conidial Cell Walls Prior to characterizing the vaccine-candidate strain, we first examined the polysaccharide structure and distribution in the wildtype control Δ akuB KU80 , the parental strain with enhanced homologous recombination used to generate the Δ sglA vaccine mutant strain. Intact, uniformly 13 C-labeled conidia were analyzed directly by solid-state NMR, while scanning electron microscopy confirmed that the cells retained the characteristic morphology of resting conidia and an intact cell–wall layer ( Figure A). In the wild-type sample, the 2D 13 C– 13 C CORD correlation spectrum acquired using dipolar-based 1 H– 13 C cross-polarization (CP), which emphasizes rigid carbohydrates, was dominated by β-1,3-glucan signals, with weaker contributions from chitin ( Figures B and S1 ). Minor signals from chitosan and β-1,6-glucan were also detected. These results reveal that the rigid core of the conidial cell wall is composed primarily of β-1,3-glucan, with additional support from chitin, chitosan, and β-1,6-glucan ( Figure C). Three magnetically distinct chitin forms were identified based on their resolved C1–C2 cross-peaks ( Figure D) and complete carbon connectivities ( Figure S2 ), reflecting local structural perturbations such as differences in conformation and hydrogen-bonding patterns. 1. Open in a new tab Rigid and mobile polysaccharides of wildtype A. fumigatus conidial cell wall. (A) Scanning electron microscopy (SEM) image of A. fumigatus dormant conidia (scale bar: 1 μm). (B) 2D 13 C– 13 C correlation spectrum of A. fumigatus conidia acquired using a 53 ms CORD experiment, which selectively detects rigid molecular components through 1 H– 13 C cross-polarization (CP). Carbon resonance assignments for chitin (Ch), β-1,3-glucan (B), α-1,3-glucan (A), chitosan (Cs), and β-1,6-glucan (H) are shown in orange, blue, green, pink, and cyan, respectively. Peak assignments are shown in the lower-right half of the spectrum (with respect to the diagonal). Each cross peak represents a correlation between two carbon atoms; for example, B1–3 corresponds to the correlation between C1 and C3 of β-1,3-glucan. (C) Summary of the NMR abbreviations used in this study, along with simplified structures of the major cell wall polysaccharides and their heterogeneous mobilities. (D) Expanded view of the 2D 13 C– 13 C correlation spectrum highlighting a zoomed-in region from Figure B that resolves three distinct chitin forms (Ch a , Ch b , and Ch c ). (E) 2D 13 C DP refocused J -INADEQUATE spectrum detecting mobile polysaccharides. Assignments contain NMR abbreviation and carbon number, for example, B1 represents β-1,3-glucan carbon 1. Glucofuranose: Gal f ; α-1,2-mannose: Mn; , α-1,6-mannose: Mn; , galactose units: Gal. (F) Selected carbohydrate regions from the 2D hcCH TOCSY (DIPSI-3) spectrum of A. fumigatus conidia, showing signals from galactofuranose (Gal f ), mannose units, glucans, and chitosan, as well as small molecules such as glucose (Glc) and galactose- or glucose-derived species (Gl). Mobile polysaccharides were identified using a combination of 13 C direct polarization and a short recycle delay in the 2D refocused J-INADEQUATE experiment, which selectively suppresses signals from rigid components with slow 13 C spin–lattice relaxation. The resulting spectrum showed well-resolved, sharp resonances from β-1,3-glucan, α-1,2-mannose, α-1,6-mannose, and galactofuranose (Gal f ) units ( Figure E). The latter three residues arise from galactomannan, whose backbone is composed of α-1,2- and α-1,6-linked mannose residues and is further substituted with β-1,5-linked (and sometimes β-1,6-linked) Gal f side chains in A. fumigatus cell walls ( Figure C). , Thus, the mobile phase of the dormant conidial cell wall consists predominantly of β-1,3-glucan and galactomannan. The identification of β-1,3-glucan in two dynamically distinct domains reveals its dual structural role, extending from the rigid inner scaffold into the mobile outer matrix and thereby bridging rigid chitin with mobile galactomannan. The mobile components also include significant contributions from proteins and lipids; however, because these species are widely distributed throughout the cell and are not specific to the cell wall, their contributions to cell wall structure were not analyzed ( Figure S3 ). Structural polymorphism was also observed among the mobile carbohydrates. Two distinct forms of Gal f and two forms of α-1,2-linked mannose were resolved in both the 13 C-detected spectrum ( Figure E) and the 1 H-detected 2D J -hcCH TOCSY spectrum ( Figure F), with 1 H detection providing enhanced sensitivity for detailed structural analysis. Notably, two Gal f forms were also detected in the mycelial cell wall, indicating that the galactomannan side chains exhibit similar structural complexity in both conidial and mycelial walls. In addition, the 2D J -hcCH TOCSY spectrum resolved three forms of β-1,3-glucan and three forms of α-1,6-linked mannose, whose complete carbon connectivities and chemical shifts were further confirmed in an extended 3D J -hCCH TOCSY data set ( Figure S4 ). This structural polymorphism reflects the molecular complexity of the soft matrix, in which polysaccharides may adopt diverse linkage and cross-linking patterns or sample multiple conformational energy minima. Deletion of sglA Alters Wall Composition and Increases α-Glucan Content and Polymorphism Compared with wildtype cells, the Δ sglA mutant displayed a slightly larger cell diameter, increasing from 1.8 to 1.9 μm ( Figure A and Table S1 ). At the molecular level, we observed a pronounced change in the rigid polysaccharide composition: the content of α-1,3-glucan increased substantially in the mutant relative to the wild type ( Figure B). This is evidenced by the enhanced α-1,3-glucan C1 and C3 peaks (A1 and A3) in the 1D 13 C CP spectra ( Figure B), as well as the appearance of strong intramolecular cross-peaks within α-1,3-glucan, such as A3-2/5, A3-4, and A3-6, in the 2D 13 C– 13 C correlation spectra ( Figure C). Peak-volume analysis indicated that the molar fraction of α-1,3-glucan in the rigid cell–wall core increased from 2% in the wild type to 20% in the mutant, accompanied by a decrease in β-1,3-glucan from 78% to 67% ( Figure D and Table S2 ). This marked shift in glucan composition suggests a reorganization of the conidial cell wall upon deletion of sglA gene, with α-1,3-glucan now providing structural integrity to the rigid cell wall, rather than a rigid matrix composed primarily of chitin/chitosan and β-glucan as in the wildtype. 2. Open in a new tab Enhanced α-glucan content, polymorphism, and altered composition in ΔsglA cell walls. (A) Cell diameter measured from SEM images. Boxes represent the interquartile range (IQR), with whiskers extending to 1.5 × IQR. Open squares indicate the mean, and horizontal lines indicate the median. Sample sizes: WT ( n = 9) and Δ sglA ( n = 9). Statistical analysis was performed using a t -test with one-tail comparison between WT and mutant strains. Statistically significant: * p -value ≤ 0.05. (B) 1D 13 C CP spectra showing rigid polysaccharides in A. fumigatus WT (top, magenta) and the Δ sglA mutant (bottom, blue). Dashed lines mark α-glucan peaks that emerge in the mutant. (C) 2D 13 C– 13 C CORD correlation spectra showing signals from rigid polysaccharides in WT (blue) and Δ sglA (magenta). (D) Molar composition of rigid polysaccharides in WT and Δ sglA , estimated from peak volume analysis of the 2D CORD spectra. (E) Identification of rigid polysaccharides using a CP-based 2D hCH experiment with a short second CP contact time (50 μs) to detect one-bond 13 C– 1 H correlations. (F) Zoomed region of the Δ sglA mutant spectrum showing distinct polymorphic forms of α-1,3-glucan. (G) Mobile molecules in WT (magenta) and Δ sglA (blue) detected by 2D 13 C-DP refocused J-INADEQUATE spectra. (H) Molar composition of mobile polysaccharides in WT and Δ sglA , analyzed from peak volumes in the 2D DP J-INADEQUATE spectra. NMR abbreviations are as follows: B, β-1,3-glucan; Ch, chitin; chitosan, Cs; A, α-1,3-glucan; GM, galactomannan; Mn, , α-1,2-mannose; Mn, , α-1,6- mannose; Gal f , galactofuranose. The 1 H-detected hCH spectra revealed that the mutant is not only enriched in α-1,3-glucan, as indicated by the increased intensity of its carbon-1 peak (A1 in Figure E), but that α-1,3-glucan also exhibits substantial structural polymorphism. This is evidenced by the broad distribution of signals for its 1 H3 and 1 H1 sites, with six magnetically nonequivalent 1 H1 environments resolved for α-glucan ( Figure F). We also observed that the combined abundance of chitin and its deacetylated form, chitosan, decreased from 16% to 8% ( Figure D). Notably, all chitosan, representing approximately 5% of the rigid molecules in the wildtype, was absent in the mutant, as indicated by the loss of the Cs4-5 cross-peak ( Figure C) and the disappearance of all characteristic chitosan carbon signals ( Figure S5 ). Thus, in the Δ sglA mutant, chitin is not only reduced in amount but also remains fully acetylated, with no detectable chitosan. Similar to the wildtype, the mutant retained a binary mobile phase composed of β-1,3-glucan and galactomannan; however, the peak intensity of β-1,3-glucan increased, leading to an increase in its molecular fraction in the mobile matrix from 22% to 46% ( Figure G,H and Table S3 ), whereas the peak intensity of galactomannan decreased substantially ( Figures G and S6 ), resulting in a reduction of its molecular fraction in the mobile matrix from 78% to 54% ( Figure H and Table S3 ). Together, these compositional and structural changes indicate a major remodeling of both the rigid core and the mobile matrix in the Δ sglA cell wall, characterized by a redistribution of a portion of β-glucan from the rigid core to the mobile phase, along with an expansion of α-1,3-glucan accompanied by reductions in chitin and galactomannan, and a complete loss of chitosan. Consolidated Δ sglA Conidial Cell Wall with Increased Rigidity, Dehydration, and Dense Packing To probe subnanometer spatial proximities between polysaccharides, we performed 2D hChH experiments with RFDR-XY16 mixing on both wild-type and mutant samples ( Figure A). This experiment produced additional intensities arising from long-range intra- and intermolecular correlations that were absent in the hCH spectrum, which primarily detects one-bond 1 H– 13 C correlations. In the wild-type sample, only a few new long-range intramolecular cross-peaks were observed, including correlations between β-1,3-glucan carbon sites and its H1 proton (B4–B H 1, B5–B H 1, and B3–B H 1), as well as between chitin C1 and its methyl proton (Ch1- H Me). In contrast, the Δ sglA mutant displayed clear intermolecular contacts ( Figure A,B). Notable cross-peaks appeared between the H1 of α-1,3-glucan and multiple β-1,3-glucan carbon sites (B4-A H 1, B2-A H 1, B5-A H 1, and B3-A H 1), as well as between α-1,3-glucan C1 and the β-1,3-glucan H1 (A1-B H 1). These interactions indicate that the abundant α-1,3-glucan uniquely observed in the mutant is effectively integrated with β-1,3-glucan on subnanoscale and has been effectively incorporated into the rigid core of the Δ sglA cell wall. These experimental observations are consistent with a potential structural role for α-1,3-glucan in providing additional structural reinforcement by acting as an adhesive molecule between various polysaccharides. 3. Open in a new tab Hydration and dynamics of polysaccharides in the A. fumigatus conidial cell wall. (A) 2D hChH spectra (0.8 ms RFDR) of WT (magenta) and Δ sglA (blue) strains. One-bond 2D hCH spectra are overlaid in black for comparison. Intermolecular cross-peaks between α-1,3- and β-1,3-glucans are underlined. (B) Schematic summary of intermolecular glucan interactions detected in Δ sglA (orange dashed lines). Arrowheads indicate polarization-transfer direction. For example, a cross-peak at C3 of β-1,3-glucan arising from the 1 H of α-1,3-glucan is labeled B3-A H 1. (C) Box-and-whisker plots of water-edited intensities ( S / S 0 ) for β-1,3-glucan (blue; n = 26, 30), α-1,3-glucan (green; n = 15, 12), and chitin (orange; n = 15, 9). Boxes show IQRs; whiskers extend to 1.5× IQR; outliers are stars. Means are open squares; medians are horizontal lines. (D) 13 C-T 1 relaxation times for β-1,3-glucan (blue; n = 5, 5), α-1,3-glucan (green; n = 3, 3), and chitin (orange; n = 3, 3). (E) 1 H-T 1ρ relaxation times for the same polysaccharides. In (D,E), magenta lines show averages across carbon sites; error bars indicate s.d. of the fit parameters into a single-exponential equation. The water-edited intensity ratios ( S / S 0 ), which report the extent of water association at individual carbohydrate sites, are reduced in the mutant relative to the wild type, indicating decreased hydration ( Figure C; Table S4 and Figures S7 and S8 ). , Among α-1,3-glucan, β-1,3-glucan, and chitin, α-1,3-glucan is the least hydrated polymer in the wild type. Because α-1,3-glucan content is increased in the mutant and shows tighter association with β-1,3-glucan, it is not surprising that the mutant cell wall becomes more dehydrated overall. The average S/S 0 values decreased from 0.81 to 0.53 for β-1,3-glucan, from 0.47 to 0.35 for α-1,3-glucan, and from 0.53 to 0.46 for chitin in the mutant relative to the wild type. In the wild-type conidia, β-1,3-glucan is the most hydrated component, bridging to the mobile phase and forming a water-rich matrix; however, in the mutant it becomes similarly dehydrated to chitin and α-glucans, likely due to its tighter association with these molecules. Together, these results suggest that the polymers in the Δ sglA cell wall adopt a more compact organization that limits water association, leading to the uniformly low hydration observed across all polymers. The molecular dynamics of the wall polymers further support this structural consolidation. Compared with the wild type, all polymers in the mutant exhibit longer relaxation time constants in both 13 C-T 1 ( Figure D) and 1 H-T 1ρ ( Figure E) measurements, indicating slower relaxation and reduced molecular motion across both the nanosecond and microsecond time scales ( Figure S9 and Table S5 ). Despite this global rigidification, the different polysaccharides display distinct dynamic behaviors intrinsically. In both samples, β-1,3-glucan shows relatively short 13 C-T 1 values but longer 1 H-T 1ρ values, consistent with rapid local motions that are nevertheless constrained at larger length scales. In contrast, α-1,3-glucan shows the opposite trend, with little flexibility at the local scale, likely due to its extensive attachment to other wall polymers, but greater motion at the microsecond time scale, reflecting slow, collective movements within the rigid network to which it is integrated. Deletion of sglA in A. fumigatus Conidia Reshapes Neutrophil Responses To better understand how the unique structural features of the Δ sglA conidial cell wall revealed by solid-state NMR influence host–pathogen interactions, we exposed A. fumigatus conidia in vitro to HL60 cells differentiated into neutrophils. We assessed both the activation state of the human cells and their ability to interact with the fungus ( Figure A–C). A modest increase in neutrophil activation was observed upon exposure to the Δ sglA mutant compared with the WT strain ( Figure A,B). More pronounced differences were detected when examining cell–fungus interactions. HL60 cells exposed to the Δ sglA mutant exhibited reduced interaction with fungal conidia, characterized by fewer adherent events and a higher proportion of fungus-free HL60 cells compared with WT exposure ( Figure C). Impaired interaction with the Δ sglA mutant diverts HL60 cells from efficient phagocytosis toward NETosis. Consequently, HL60 cells exposed to the Δ sglA mutant exhibit markedly increased NET formation ( Figure D), reduced fungal killing capacity ( Figure E), and decreased cell viability ( Figure F). Thus, SG accumulation and cell wall remodeling in the Δ sglA mutant lead to an increased capacity to trigger immune activation in HL60 cells in vitro. 4. Open in a new tab Effect of A. fumigatus sglA deletion on neutrophil response in vitro and in vivo. (A) Neutrophil-like differentiated HL60 cells either not-treated (NT) or stimulated with 1:1 ratio of A. fumigatus conidia (WT or Δ sglA ) for 2 h at 37 °C. After stimulation, cells were cytospin and stained with May-Grünwald Giemsa. (B) Cell morphology and (C) interaction with Aspergillus conidia were evaluated. Nonactivated cells were round; activated cells displayed an irregular shape and a reduced cytoplasm-to-nucleus ratio; highly activated cells exhibited blebbing and long cytoplasmic protrusions. Cells were considered dead when appearing small and anucleated. Conidia-cell interactions were quantified as the percentage of free (unbound), adherent (surface-associated), or internalized (phagocytosed) conidia. Statistical significance was determined by two-way ANOVA (* P < 0.05, ** P < 0.01, **** P < 0.0001). (D) Frequency of NETosis events. Statistical significance was determined by one-way ANOVA (** P < 0.01). (E) Ability of neutrophil-like differentiated HL60 to eliminate Aspergillus conidia after 2 h stimulation assessed with a killing assay. (F) Vitality of neutrophil-like differentiated HL60 after 24 h stimulation with conidia at 1:1 ratio, expressed as percentage of the nontreated controls. (G) A murine model of invasive aspergillosis established through intranasal injection of 6 × 10 7 WT or Δ sglA Aspergillus conidia for three consecutive days. (H) Lung CFUs counted after mice were sacrificed on day-7. (I) Bronchioalveolar lavage (BAL) recovered and fixed trough cytospin and stained with May-Grunwald Giemsa. (J) Neutrophil numbers in BAL counted and expressed as percentage of total cells. (K) Elisa test for IFN-γ, IL-12, TNF-α, IL-27, IL-17A and IL-23 performed on the lung homogenate of sacrificed mice. Statistical significance was determined by unpaired t -test (* P < 0.05, ** P < 0.01). Next, we examined the impact of this altered interaction in vivo, using an immunocompetent mouse model of invasive aspergillosis ( Figure G), thereby avoiding converting mice into immunodeficient, to assess the effect on neutrophilic response in vivo. At 7 days postinfection, mice exposed to the Δ sglA mutant exhibited significantly higher lung fungal burden and increased neutrophil recruitment compared with WT-infected mice ( Figure H–J). Despite these differences, overall survival did not differ between WT- and Δ sglA -infected mice. When measuring cytokine production in the lungs of Δ sglA -infected mice, we found a significant decrease in cytokines usually produced upon Dectin-1 interaction, including IL-27, IL-23, IL-17A, and TNF-α, compared with WT-infected mice ( Figure K). In contrast, IL-12 levels were significantly higher in Δ sglA -infected mice compared with WT-infected mice ( Figure K), consistent with increased engagement of the mutant strain with Toll-like receptor 2 (TLR2). Discussion In this study, we show that deletion of sglA gene in A. fumigatus conidia triggers extensive remodeling of the cell wall, characterized by (i) a marked increase in α-1,3-glucan within the rigid inner scaffold, (ii) a compensatory decrease in β-1,3-glucan in this compartment, (iii) reduced amount of chitin and a complete loss of chitosan, (iv) enhanced intermolecular contacts between α-1,3- and β-1,3-glucans, (v) restricted molecular motions, and (vi) reduced water accessibility. Together, these features may help preserve cell wall strength and integrity in the absence of sglA -dependent homeostasis. This tightened conidial cell–wall architecture plausibly contributes to the altered phenotype, in which Δ sglA conidia exhibit reduced germination capacity, shorter germ tubes, and delayed hyphal growth. These structural features can be understood in the broader context of fungal cell wall organization and remodeling. Notably, the reduction in chitin observed here in intact resting conidia is consistent with recent chemical analyses of Δ sglA cell–wall carbohydrates. In that earlier study, β-glucan levels appeared unchanged relative to the wildtype; however, our present data refine this interpretation by showing that β-glucan is undergoing a phase redistribution, with a fraction shifting from the rigid scaffold to the more mobile matrix. It is also important to note that the previous measurements were performed on mixed morphotypes, including germinating conidia, hyphae, and conidia that remained ungerminated, whereas the current work focuses exclusively on resting conidia. These complementary data sets suggest that β-glucan remodeling is stage-dependent, varying across distinct phases of fungal differentiation. It should also be noted that conventional mass spectrometry- and chromatography-based compositional analyses of alkali-soluble and alkali-insoluble fractions of the fungal cell wall typically rely on extraction and hydrolysis of cell–wall polysaccharides prior to measurement, procedures that can disrupt polymer structure. In contrast, solid-state NMR probes intact cells in situ, enabling comparison of polysaccharide signals while simultaneously reporting polymer mobility, hydration, and intermolecular associations. Two recent studies of A. fumigatus cell walls combining biochemical and solid-state NMR analyses have reported generally comparable compositional trends using both approaches, although the absolute values are not strictly identical due to differences between the two techniques. , Therefore, the NMR-based measurements complement biochemical approaches by providing compositional information together with structural context for polymer organization within the cell wall. Recently, solid-state NMR and functional-genomics studies of A. fumigatus hyphae have defined a general architectural framework for the fungal cell wall, in which a rigid scaffold of chitin, β-1,3-glucan and α-1,3-glucan is embedded within a more mobile matrix enriched in galactomannan, glucans, and other biopolymers, such as galactosaminogalactan. ,, This bimodal organization is not static: under environmental and pharmacological stress, the wall undergoes coordinated remodeling in which the relative abundance and interactions of these polymers are dynamically rebalanced to preserve integrity. ,, These observations establish a key conceptual principle: the cell wall functions as a reconfigurable polysaccharide composite whose rigid core can be reinforced or redistributed in response to perturbation. Building on this structural framework, conidial cell walls represent a developmentally specialized form of this architectural system. Solid-state NMR snapshots across morphotype transitions show that conidia possess a rigid, relatively dehydrated inner scaffold that undergoes defined polymer reorganization at the onset of germination. Dormant conidia have a rigid core in which β-1,3-glucan contributes more strongly than α-1,3-glucan and chitin, whereas during swelling the amount of α-1,3-glucan temporarily doubles before returning toward its original proportion as germination progresses. Developmental transitions are accompanied by increased surface accessibility of wall polysaccharides, including exposure of α-1,3-glucan during swelling and the appearance of mobile galactosaminogalactan at the surface of emerging germ tubes, thereby linking these early remodeling events to the structural principles that support the formation of mature hyphae at later developmental stages. Consistent with this developmental specialization, Kre6-dependent β-1,6-glucan biosynthesis appears to be restricted to the conidial stage, reinforcing the view that polysaccharide composition and wall architecture are developmentally programmed and reshaped during the transition to germination. − Across these contexts, α-1,3-glucan is emerging as a central adhesive and buffering polymer. Although early studies suggested that α-1,3-glucan was dispensable, as synthase deletions caused only subtle growth or virulence phenotypes, solid-state NMR later revealed that α-1,3-glucan is a major structural component that packs with chitin and β-1,3-glucan to form the stiff inner cell–wall core in A. fumigatus . , This supports a model in which α-1,3-glucan physically stabilizes interactions among wall polysaccharides and buffers architectural changes during morphogenesis and stress-induced remodeling. ,, Its adhesive role also provides a mechanistic explanation for earlier observations that surface-exposed α-1,3-glucan mediates aggregation of swelling conidia, an effect that will be abolished by treatment with α-1,3-glucanase. , The structural role of α-1,3-glucan in cell–wall construction is also evident in other fungi, such as C. neoformans , where it makes up the bulk of the rigid cell–wall core and interacts with essentially all other polysaccharides, along with melanin and the capsule. Previous in vivo studies demonstrated that Δ sglA conidia are efficiently cleared from immunosuppressed hosts while conferring complete protection against subsequent lethal wild-type challenge, in both live and heat-killed form. In this study, the in vitro observations of reduced neutrophil-fungus interaction, impaired phagocytosis, diversion toward NETosis, and decreased fungal killing, along with the in vivo evidence of increased fungal burden, heightened neutrophil recruitment, and altered cytokine production, are consistent with a model in which cell wall remodeling in the Δ sglA mutant alters pathogen-associated molecular pattern (PAMP) exposure and disrupts normal host immune recognition. Consistent with this interpretation, and at the molecular level, solid-state NMR analysis confirmed that the Δ sglA mutant exhibits a substantial increase in α-1,3-glucan content, including magnetically distinct α-1,3-glucan polymorphs that show extensive interactions with β-glucans. This expanded and well-integrated α-1,3-glucan fraction likely restricts and masks the associated β-1,3-glucan, reducing its accessibility to Dectin-1 during swelling, a stage at which β-1,3-glucan normally becomes exposed and recognized by innate immune cells, and thereby weakening β-glucan-dependent antifungal responses while redirecting immune signaling toward alternative pathways such as TLR2. As reported for other opportunistic fungal pathogens, the presentation of α-1,3-glucan can effectively conceal β-1,3-glucan signatures and disrupt normal host immune recognition. − In this way, structural reorganization of the Δ sglA cell wall may contribute to the altered immune recognition and neutrophil responses observed in both cellular and animal models. Although the β-glucan content decreased in the rigid core, it remains plausible that the reduced Dectin-1 activity we observed occurred even though the β-glucan content increased in the mobile phase. This suggests that (i) the mobile phase detected here does not necessarily correspond to the outer cell–wall layer, but instead reflects portions of the mobile matrix that neither aggregate nor associate with chitin; and (ii) the mobile β-1,3-glucan is not responsible for Dectin-1 binding. Rather, only a subset of the more rigid β-1,3-glucan adopts the appropriate conformation for Dectin-1 recognition, consistent with reports that pattern-recognition receptors such as Dectin-1 bind most effectively to the triple-helical structure of β-glucan. − The combined reduction of triple-helical β-1,3-glucan in the rigid domain, conformational changes in the mobile domain, and enhanced α-1,3-glucan shielding are expected to reduce β-1,3-glucan accessibility to immune receptors, thereby altering immune recognition of A. fumigatus . Together, these observations provide a plausible explanation for the reduced host immune response. Future work should correlate NMR-observed structural polymorphism with the diverse functional and immunological roles of cell–wall carbohydrates. The adaptive remodeling mechanisms observed here differ from those triggered by antifungal treatment. For example, caspofungin inhibits β-1,3-glucan synthesis and reduces its abundance in both the rigid and mobile wall domains of the A. fumigatus cell wall. In contrast, deletion of sglA does not eliminate β-1,3-glucan but redistributes it, decreasing its contribution to the rigid scaffold while increasing its relative fraction in the mobile matrix. Despite these differences, both perturbations induce compensatory cell wall reorganization. In caspofungin-treated cultures, reduced β-1,3-glucan is accompanied by increased α-1,3-glucan, chitin, and chitosan. In the Δ sglA mutant, loss of rigid-domain β-1,3-glucan is associated with enrichment of α-1,3-glucan, reduced chitin, and complete loss of chitosan. Notably, increased α-1,3-glucan emerges as a common adaptive response in both contexts. Consistent with these structural changes, caspofungin treatment impairs hyphal growth, whereas the Δ sglA mutant shows delayed hyphal development. These results reveal that perturbation of β-1,3-glucan biosynthesis and structure, whether by enzymatic inhibition or genetic mutation, exposes a structural vulnerability that triggers coordinated polysaccharide remodeling of the A. fumigatus cell wall. Taken together, our results support a model in which α-1,3-glucan acts both as an architectural adhesive and as a compensatory structural buffer in the Δ sglA mutant, stabilizing the rigid scaffold while retaining a substantial fraction of β-1,3-glucan through intermolecular interactions and thereby limiting its accessibility to host receptors during swelling. This organization provides a structural basis for the immunoprotective properties of the Δ sglA strain and parallels observations in C. neoformans , where related mutants show altered engagement of pattern-recognition receptors. , Future studies dissecting receptor-specific signaling will be important to define how these architectural changes shape the immune–protective profile of Δ sglA and related vaccine candidates. Materials and Method Preparation of Uniformly 13 C, 15 N-Labeled A. fumigatus Cells for NMR Analysis Two A. fumigatus strains were used: the parental strain Δ akuB KU80 , which enhances homologous recombination, and the Δ sglA mutant, which has shown potential as a vaccine candidate. The strains were cultured on agar plates containing 20 g/L 13 C-glucose (Catalog # CLM-1396-PK, Cambridge Isotope Laboratories) and a sodium nitrate salt solution (NLM-712-PK, Cambridge Isotope Laboratories) as the sole carbon and nitrogen sources, respectively, and supplemented with trace elements ( Table S6 ). Cultures were incubated at 37 °C for 3 days. Conidia were collected from the plates using an aqueous 0.5% Tween-20 solution, washed twice with deionized water, followed by a wash with phosphate-buffered saline (PBS) to remove excess salts and glucose, and then centrifuged at 3000 rpm for 10 min. The intact conidia were packed into a 3.2 mm rotor or a 1.3 mm rotor for solid-state NMR analysis. SEM Imaging of Cell Morphology Fungal cultures were harvested and fixed in 4% (v/v) glutaraldehyde prepared in 0.1 M sodium phosphate buffer (pH 7.4) for 1–2 h at 4 °C. Following primary fixation, samples were rinsed three times with PBS and postfixed in 1% (w/v) osmium tetroxide for 1–2 h at room temperature. Specimens were then dehydrated through a graded ethanol series (25%, 50%, 75%, and 95%), with each dehydration step carried out for 10–15 min. Dehydrated samples were subjected to critical point drying using a Leica EM CPD300 with CO 2 as the transitional fluid. The dried material was mounted onto aluminum stubs using conductive carbon tape. SEM was performed using a JEOL JSM-7500F field-emission instrument, and micrographs were obtained at multiple magnifications to evaluate fungal surface morphology. 13 C Solid-State NMR Analysis of Polysaccharides Present in Conidial Cell Wall High-resolution solid-state NMR spectroscopy was performed on a Bruker Avance Neo 800 MHz spectrometer equipped with a 3.2 mm HCN triple-resonance MAS probe at the Max T. Rogers NMR Facility, Michigan State University. 13 C-detected experiments were carried out at a magic-angle spinning (MAS) frequency of 15 kHz and a regulated sample temperature of 298 K. Chemical shifts were externally referenced to the tetramethylsilane (TMS) scale by calibrating the methylene (CH 2 ) resonance of adamantane to 38.48 ppm. Radiofrequency (rf) field strengths for 1 H hard pulses, heteronuclear decoupling, and CP transfers ranged from 71.4 to 83.3 kHz. 13 C pulses were applied using radiofrequency field strengths of 50 or 62.5 kHz, depending on the specific experiment. Resonance assignments of carbon sites within polysaccharides were obtained using a series of 2D solid-state NMR experiments designed to probe the molecular organization of the fungal cell wall. Through-bond 13 C– 13 C connectivities of mobile components were characterized using 13 C-DP refocused J-INADEQUATE experiments carried out with a short 2-s recycle delay. , The J-evolution period consisted of four delays of 2.3 ms each, optimized for achieving the highest carbohydrate intensity. In parallel, rigid polysaccharides were analyzed using CP-based 2D correlation experiment using a 53 ms CORD (COmbined R2nv-Driven) mixing period at 13.5 kHz MAS ( Figure S2 ). , The 13 C signals of mobile and rigid carbohydrates were assigned based on carbon connectivities in the J-INADEQUATE spectra and intramolecular cross-peaks in the CORD spectra, and were cross-validated using data from the Complex Carbohydrate Magnetic Resonance Database and recent literature reports. The chemicals shifts are documented in Table S7 . Compositional Analysis of Cell Wall Carbohydrates Analysis of peak volumes from 2D 13 C-CP CORD and 2D 13 C-DP refocused J-INADEQUATE spectra was used to estimate the relative molar composition of rigid and mobile carbohydrates in each sample, respectively ( Tables S2 and S3 ). Peak volumes were integrated using the Bruker TopSpin software package (version 4.1.4). To minimize errors arising from spectral overlap, only well-resolved and unambiguous resonances were included in the quantitative analysis. For the CORD spectra, analysis was performed by averaging the volumes of clearly resolved cross-peaks associated with each polysaccharide component. For the INADEQUATE spectra, only well-defined scalar-coupled carbon pairs were considered. All spectra compared across samples were acquired and processed using identical pulse sequences, acquisition parameters, and processing conditions, and the data were normalized by the number of scans. Relative molar abundances were calculated by normalizing the integrated peak volumes to the number of contributing resonances for each carbohydrate species, and the resulting values were expressed as fractions of the total carbohydrate signal within the corresponding spectral region. These values therefore represent estimated relative molecular fractions within the rigid and mobile carbohydrate matrices rather than absolute concentrations. Standard errors were estimated by dividing the standard deviation of the integrated volumes by the number of cross-peaks included in the analysis. Total standard error for each sample was obtained by calculating the square root of the sum of the squared individual errors as described recently. Profiling the Hydration and Mobility of Cell Wall Polysaccharides The dynamics of cell wall polysaccharides were analyzed using two relaxation methods. The 13 C-T 1 relaxation times were measured using the Torchia-CP pulse sequence with z-filter durations ranging from 0.1 μs to 12 s. For each resolved resonance, the decay in signal intensity was monitored as the z-filter duration increased, and the resulting curves were fit to a single-exponential function to obtain the 13 C-T 1 relaxation time constants. Absolute intensities were prenormalized to the number of scans collected for each spectrum. Similarly, 13 C-detected 1 H-T 1ρ relaxation times were measured using a Lee-Goldburg (LG) spinlock with varying duration combined with LG-CP. This approach effectively suppressed 1 H– 1 H spin diffusion during both the spinlock and CP periods, enabling site-specific determination of 1 H-T 1ρ relaxation by detecting the directly bonded 13 C sites. Peak intensity decays were modeled using a single-exponential function to extract the 1 H-T 1ρ time constants. All relaxation data sets were processed and analyzed using Origin 2021. To probe polysaccharide hydration levels, water-edited 2D 13 C– 13 C correlation spectra were acquired. ,,, The experiment began with 1 H excitation, followed by a 1 H-T 2 filter (0.45 ms × 2 for the WT sample and 0.6 ms × 2 for the mutant), which eliminated 97% of polysaccharide proton signals while retaining 84% of bulk water magnetization. Water-derived magnetization was then transferred to polysaccharides using a 4 ms 1 H mixing step, followed by a 1 ms 1 H– 13 C CP period for site-specific 13 C detection. A 50 ms DARR mixing period was applied to both the water-edited spectrum and a corresponding control 2D spectrum that preserved full signal intensity. Hydration levels were quantified by calculating the relative intensity ratios between the water-edited ( S ) and control ( S 0 ) spectra for all cell wall samples. Signal intensities were normalized to the number of scans collected for each data set before analysis. The key experimental parameters are summarized in Table S8 . 1 H-Detected Solid-State NMR Resolving Polymorphism and Intermolecular Interactions Rigid molecules in A. fumigatus cell walls were characterized using CP-based 1 H-detected experiments on a Bruker Avance Neo 600 MHz spectrometer located at the Max T. Rogers NMR Facility at Michigan State University with a fast-MAS 1.3 mm HCN triple-resonance probe spinning at 60 kHz. 13 C chemical shifts were externally referenced to the TMS scale, and 1 H chemical shifts were referenced to the DSS scale. Rigid polysaccharide regions were investigated using two 1 H-detected experiments, including 2D hCH and 2D hChH with RFDR-XY16 mixing. ,, One-bond 13 C– 1 H correlations were obtained using the 2D hCH experiment via a short second CP contact time of 50 μs, while through–space correlations were generated using the 2D hChH experiment via a 1 H– 1 H RFDR-XY16 homonuclear dipolar recoupling period with a mixing time of 0.533 ms. The 90° pulse lengths were 2.5 μs (100 kHz) for 1 H and 4 μs (62.5 kHz) for 13 C. slpTPPM (swept low-power two-pulse phase modulation) decoupling was applied on the 1 H channel during t 1 evolution, with a radiofrequency field strength of 12.8 kHz, and WALTZ-16 decoupling was applied on 13 C during proton detection at 20.2 kHz. Water suppression was achieved using the MISISSIPPI sequence (16 kHz, 100 ms). For both experiments, 448 TD points were acquired with 32 scans per increment and a recycle delay of 2 s, resulting in total acquisition times of 8 h 34 min (hCH) and 8 h 28 min (hChH). All multidimensional data sets were collected using the States-TPPI method. Detailed experimental parameters and assigned 13 C and 1 H chemical shifts are provided in Tables S9 and S10 . Mobile regions of the A. fumigatus conidial cell walls were investigated using J-coupling-based proton-detected experiments on a Bruker Avance Neo 800 MHz spectrometer equipped with a 3.2 mm HCN triple-resonance MAS probe operating at 15 kHz. The mobile molecules do not require fast MAS, as their intrinsic dynamics average out a significant portion of the 1 H– 1 H dipolar coupling. Mobile polysaccharides were assigned using a 2D 13 C– 1 H correlation experiment J-hCcH TOCSY (total correlation spectroscopy) and 3D 13 C– 13 C– 1 H correlation experiment J-hCCH TOCSY employing DIPSI-3 (decoupling in the presence of scalar interactions) mixing. , The 90° pulse widths were set to 3.5 μs (71.4 kHz) for 1 H and 5.0 μs (50 kHz) for 13 C. SPINAL-64 (small phase incremental alternation with 64 steps) heteronuclear decoupling was applied during the t 1 and t 2 evolution periods with an rf field strength of 71.4 kHz, while WALTZ-16 (wideband alternating-phase low-power technique for zero-residual splitting) decoupling was applied on 13 C during 1 H detection with an radiofrequency field strength of 17 kHz. , Water suppression was achieved using the MISSISSIPPI (multiple intense solvent suppression intended for sensitive spectroscopic investigation of protonated proteins) sequence with a 26 kHz radiofrequency field applied for 40 ms. Broadband DIPSI-3 mixing was employed to obtain 13 C– 13 C correlations using a 2 ms spin-lock pulse. The mixing time was set to 25.5 ms, with a radiofrequency field strength of 17 kHz applied for both the DIPSI-3 and spin-lock pulses. For the wild-type A. fumigatus conidial sample, the 3D hCCH TOCSY experiment was acquired with 128 time-domain (TD) points in both the t 1 and t 2 dimensions. Eight transients were coadded per TD point using a recycle delay of 2 s, resulting in a total experimental time of 3 days, 5 h, and 53 min. The 2D hcCH TOCSY experiment with DIPSI-3 mixing was performed on both samples using the same parameters as the 3D experiment, except t 1 was set to a single TD point and t 2 to 512 TD points. Thirty-two scans were acquired per increment with a recycle delay of 2 s, yielding a total experiment time of 9.6 h ( Tables S11 and S12 ). HL60 Culture and Differentiation After thawing, HL60 were cultured into tissue-treated flasks maintaining a concentration between 1 × 10 5 and 5 × 10 5 cell/mL splitting every 2 to 3 days. The culture medium consisted of RPMI supplemented with 10% fetal bovine serum (FBS), 1% penicillin–streptomycin, and 1% GlutaMAX (Invitrogen). For differentiation into neutrophil-like HL60 cells, cells were stimulated by adding 1.25% DMSO to the culture medium for 7 to 10 days. To check differentiation status, cytofluorimetry was performed to measure increased expression of surface marker CD16b compared to nontreated HL60. HL60 and Aspergillus Interaction Assay Neutrophil-like HL60 cells (5 × 10 5 ) were incubated in 1 mL of culture medium either without stimulus or with A. fumigatus conidia at a 1:1 cell-to-conidium ratio for 2 h at 37 °C 5% CO 2 . Following incubation, 50 μL of cell suspension was recovered, and cells were cytospinned into microscopy slides through a cytospin chamber at 700 × g for 7 min and minimum rotor acceleration and deceleration. Produced slides were stained with May-Grunwald-Giemsa staining (Sigma-Aldrich). Cell morphology, interaction between cells and conidia and netosis events were measured, counting 10 fields at 60-fold magnification for each slide on an EVOS FL Auto Imaging System (Thermo-Fisher Scientific). Cells undergoing NETosis were distinguished by the presence of densely Giemsa-stained smears. HL60 Vitality Assay Neutrophil-like HL60 cells (5 × 10 5 ) were incubated in 1 mL of culture medium either without stimulus or with A. fumigatus conidia at a ratio of 1:1 for 24 h at 37 °C 5% CO 2 . After the incubation, vitality was assessed using trypan blue solution and a hemocytometer. Aspergillus Killing Assay Conidia of A. fumigatus (5 × 10 5 ), both wild-type and Δ sglA , were incubated in 100 μL of culture medium for 2 h at 37 °C 5% CO 2 with and without neutrophil-like HL60 cells at a ratio of 1:1. Following incubation, 10 μL of TRITON 100X was added to each well and mixed vigorously. The plate was left to incubate for 15 min at 37 °C to lyse the HL60 cells. Each well was diluted 1 to 5000 into PBS/tween20 0.05%. 100 μL of each solution was seeded in Sabouraud agar plates (Merk Millipore) and left to incubate overnight at 37 °C. A. fumigatus CFUs were counted, and the killing percentage was expressed as the difference between conidia not exposed to phagocytic cells and those exposed. Murine Model of Invasive Aspergillosis Experimental protocols for murine in vivo studies were approved by the Ministry of Health (Authorization N. 310/2025-PR) and previously certified by the animal ethics committee ‘OPBA’ from the University of Perugia, Italy. All mice used in this study were female C57BL/6 mice, 8–10 weeks old, purchased from Charles River Mice were anesthetized by intraperitoneal (i.p.) injection of 2.5% Avertin (Sigma Chemical Co.) before intranasal instillation of 6 × 10 7 A. fumigatus resting conidia suspended in 20 μL of saline, administered once daily for three consecutive days. Mice were euthanized on day 7. Bronchioalveolar lavage (BAL) was performed on sacrificed animals by cannulating the trachea and washing the airways with PBS to collect the BAL fluid. Differential cell counts were generated on BAL smears stained with May-Grünwald Giemsa (Sigma-Aldrich) reagents, counting 10 fields at 60× magnification on an EVOS FL Auto Imaging System (Thermo-Fisher Scientific). Lungs recovered from sacrificed mice and homogenized in 1 mL of PBS. 100 μL of homogenate was seeded in Sabouraud agar plates (Sigma-Aldrich) and incubated at 37 °C overnight for CFU counting. Homogenate was centrifuged at 2000 rpm for 10 min, and supernatant was recovered for ELISA test. ELISA tests for IFN-γ (Biolegend), IL-12 p40 (eBioscience), TNF-α, IL-27, IL-17A, IL-23 p19 (Invitrogen) were performed as per the producers’ instructions. Supplementary Material ja6c00915_si_001.pdf (2.1MB, pdf) Acknowledgments The research was supported by the National Institute of Health (NIH) under award numbers R01AI173270 to T.W. and R01AI125770 to M.D.P. Glossary Abbreviations 3D hCCH-TOCSY total correlation spectroscopy CORD combined R2nν-driven CP cross-polarization DIPSI-3 decoupling in the presence of scalar interactions DP direct polarization DSS sodium trimethylsilylpropanesulfonate FBS fetal bovine serum Gal f galactofuranose INADEQUATE Incredible natural abundance double quantum transfer experiment LG Lee-Goldburg MISSISSIPPI multiple intense solvent suppression intended for sensitive spectroscopic investigation of protonated proteins PAMP pathogen-associated molecular pattern PBS phosphate-buffered saline RFDR-XY16 radio frequency-driven recoupling SEM scanning electron microscopy slpTPPM swept low-power two-pulse phase modulation SPINAL-64 small phase incremental alternation with 64 steps TD time-domain TLR2 Toll-like receptor 2 TMS tetramethylsilane TOCSY total correlation spectroscopy WALTZ-16 wideband alternating-phase low-power technique for zero-residual splitting All relevant data that support the findings of this study are provided in the article and Supporting Information . All the original ssNMR data files, pulse sequences, and experimental parameters are deposited in the Zenodo repository with the DOI for public access: 10.5281/zenodo.18877267 . The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.6c00915 . Additional NMR spectra, analysis, experimental parameters, and chemical shifts are documented in Figures S1–S9, Tables S1–S12, and Supplementary References ( PDF ) #. K.S. and A.A. contributed equally. The authors declare the following competing financial interest(s): Dr. Maurizio Del Poeta, M.D., is a Co-Founder and Chief Scientific Officer (CSO) of MicroRid Technologies Inc. The goal of MicroRid Technologies Inc. is to develop new antifungal agents for therapeutic use. All other authors declare no competing interests. References Brown G. D., Denning D. W., Gow N. A. R., Levitz S. M., Netea M. G., White T. C.. Hidden killers: human fungal infections. Sci. Transl. 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Supplementary Materials ja6c00915_si_001.pdf (2.1MB, pdf) Data Availability Statement All relevant data that support the findings of this study are provided in the article and Supporting Information . All the original ssNMR data files, pulse sequences, and experimental parameters are deposited in the Zenodo repository with the DOI for public access: 10.5281/zenodo.18877267 . 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