Molecular architecture of fungal cell walls revealed by solid-state NMR

Abstract

The high mortality of invasive fungal infections, and the limited number and inefficacy of antifungals necessitate the development of new agents with novel mechanisms and targets. The fungal cell wall is a promising target as it contains polysaccharides absent in humans, however, its molecular structure remains elusive. Here we report the architecture of the cell walls in the pathogenic fungus Aspergillus fumigatus. Solid-state NMR spectroscopy, assisted by dynamic nuclear polarization and glycosyl linkage analysis, reveals that chitin and α-1,3-glucan build a hydrophobic scaffold that is surrounded by a hydrated matrix of diversely linked β-glucans and capped by a dynamic layer of glycoproteins and α-1,3-glucan. The two-domain distribution of α-1,3-glucans signifies the dual functions of this molecule: contributing to cell wall rigidity and fungal virulence. This study provides a high-resolution model of fungal cell walls and serves as the basis for assessing drug response to promote the development of wall-targeted antifungals.

Fig. 1

Chitin and glucans form the rigid domain of intact A. fumigatus cell walls. a 2D ¹³C–¹³C correlation spectrum measured with 53-ms CORD mixing detects all intramolecular cross peaks of chitin and four types of glucans. Abbreviations are used for resonance assignment and different polysaccharide signals are color coded. b ¹³C CP J-INADEQUATE spectrum resolves the ¹³C through-bond connectivity for each polysaccharide. c Identified polysaccharides and representative chemical shifts. All spectra were measured on an 800 MHz solid-state NMR spectrometer

Fig. 2

Chitin is structurally polymorphic in intact A. fumigatus cell walls. a 1D ¹⁵N spectrum resolved multiple amide and amine signals. b High-resolution ¹⁵N–¹³C correlations spectra resolved three major types of chitins. c DNP-assisted ¹⁵N–¹⁵N PAR spectra measured using 5 ms (red) and 15 ms (black) mixing times revealed extensive cross peaks between different chitin allomorphs. d DNP-assisted ¹⁵N–¹³C correlation spectra measured using 3-mg A. fumigatus

Fig. 3

MAS-DNP solid-state NMR reveals the tight packing of chitin and α-1,3-glucan. a 15 ms ¹³C–¹³C PAR spectrum (black) reports many intermolecular cross peaks, mainly between chitin and α-1,3-glucans. A 100-ms DARR spectrum (orange) that primarily detects intramolecular correlations is overlaid for comparison. b Summary of 65 long-range restraints. For each category, the number of all cross peaks and the number of strong and intermediate restraints (in parenthesis) are listed. c Sensitivity enhancement ε_on/off of 30-fold was obtained for A. fumigatus. A picture of a DNP sample is also included. d DNP-assisted intermolecular-only ¹⁵N–¹³C correlation spectrum unambiguously detected several chitin–glucan cross peaks. e DNP-assisted chitin-edited spectrum only shows signals from chitin itself or the glucans that are spatially proximal. The DNP experiments were conducted on a 600 MHz/395 GHz spectrometer

Fig. 4

Chitin and α-1,3-glucans form the hydrophobic core of A. fumigatus cell walls. a Overlay of 2D water-edited (red) and control (black) ¹³C–¹³C correlation spectra and b 1D ¹³C cross sections. c Relative intensities of different polysaccharides in water-edited spectra. The water-edited spectrum preferentially detects well-hydrated molecules. Error bars indicate standard deviations propagated from NMR signal-to-noise ratios. Chitin and α-1,3-glucans have the lowest intensities in water-edited spectra (shaded)

Fig. 5

A. fumigatus cell walls are dynamically heterogeneous. a 1D ¹³C DP spectra of intact A. fumigatus cells with quantitative detection of all molecules (black) or selective detection of the mobile components (red). Abbreviations of carbohydrate names, carbon numbers, and subtypes (superscripts) are included in the assignment. b Difference spectrum obtained by subtraction of the two 1D DP spectra. c ¹³C CP spectrum that favors rigid molecules. d Peak intensity ratios between different DP spectra and the CP spectrum show that α-1,3-glucan and a subset of chitins are relatively rigid. Error bars are standard deviations propagated from NMR signal-to-noise ratios. e ¹H-T_1ρ relaxation times. The open and filled bars represent the short and long-components of ¹H-T_1ρ relaxation. Error bars are standard deviations of the fit parameters. Dashlines indicate the average value of the longer ¹H-T_1ρ component for each type of polysaccharides

Fig. 6

Glycoprotein and α-1,3-glucan form a highly mobile shell. a 2D ¹³C DP J-INADEQUATE spectrum selects only the very mobile molecules of proteins and polysaccharides. b The polysaccharide region reveals the presence of β-1,4-mannan, arabinan and α-1,3-glucan

Fig. 7

Illustrative model of the supramolecular architecture of A. fumigatus cell walls. Dashline circles highlight the intermolecular interactions with the total numbers of NMR restraints indicated. The number of strong restraints is in parenthesis. The average hydration levels (percentage values) and the average ¹H-T_1ρ relaxation times (in millisecond) of each polymer are also labeled