Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy

Abstract

Aspergillus fumigatus is commonly responsible for lethal fungal infections among immunosuppressed individuals. A. fumigatus forms biofilm communities that are of increasing biomedical interest due to the association of biofilms with chronic infections and their increased resistance to antifungal agents and host immune factors. Understanding the composition of microbial biofilms and the extracellular matrix is important to understanding function and, ultimately, to developing strategies to inhibit biofilm formation. We implemented a solid-state nuclear magnetic resonance (NMR) approach to define compositional parameters of the A. fumigatus extracellular matrix (ECM) when biofilms are formed in RPMI 1640 nutrient medium. Whole biofilm and isolated matrix networks were also characterized by electron microscopy, and matrix proteins were identified through protein gel analysis. The (13)C NMR results defined and quantified the carbon contributions in the insoluble ECM, including carbonyls, aromatic carbons, polysaccharide carbons (anomeric and nonanomerics), aliphatics, etc. Additional (15)N and (31)P NMR spectra permitted more specific annotation of the carbon pools according to C-N and C-P couplings. Together these data show that the A. fumigatus ECM produced under these growth conditions contains approximately 40% protein, 43% polysaccharide, 3% aromatic-containing components, and up to 14% lipid. These fundamental chemical parameters are needed to consider the relationships between composition and function in the A. fumigatus ECM and will enable future comparisons with other organisms and with A. fumigatus grown under alternate conditions.

FIG 1

Electron microscopy of A. fumigatus biofilm preparations and ECM protein analysis. (A to C) Scanning electron micrographs of intact A. fumigatus biofilm. (A) This lower-magnification micrograph shows the collection of hyphae associated with the A. fumigatus biofilm formed in the modified version of RPMI medium used in this study. ECM connective material is present between some of the hyphae (indicated by red arrows). (B) At higher magnification, wide sheets of ECM are seen stretching between hyphae. (C) ECM that is closely associated with hyphae can be observed at the highest resolution, with an apparent rough and web-like coating. (D) Transmission electron micrograph of isolated A. fumigatus ECM. Fibrous material is indicated by a red arrow, and vesicle-type structures are also apparent. (E) SDS-PAGE protein gel of the A. fumigatus ECM. Five representative bands of the SDS-soluble proteins (labeled 1 to 5) were excised and identified. Catalase B (band 1, 79,910 Da) and Asp f2 (band 2, 32, 838 Da) were specifically identified. Bands 3 to 5 corresponded to processed forms of these two proteins.

FIG 2

Spectral comparisons of the carbon and nitrogen composition of the A. fumigatus biofilm and isolated ECM. The ¹³C CPMAS spectrum of the A. fumigatus intact biofilm sample (cells plus ECM) contains major contributions from polysaccharides, consistent with the thick hyphal cell walls (45,000 scans). The ¹³C CPMAS spectrum of the ECM is consistent with a material containing proteins (carbonyl and alpha carbon peaks), aromatic compounds (such as melanin), and polysaccharides (48,000 scans). The spinning side bands, which are a result of the magic-angle spinning and not representative of a specific carbon type, are indicated by asterisks. The ¹⁵N CPMAS spectrum of the whole biofilm contains a prominent peak centered at 122 ppm that is indicative of amides. The smaller peaks are indicative of nitrogen-containing amino acid side chains (12,000 scans). The ¹⁵N CPMAS spectrum of the ECM contains a similarly prominent amide peak centered at 119 ppm plus other smaller peaks and nitrogen contributions (12,000 scans).

FIG 3

Two-sample comparison of ECM carbon contributions. ¹³C CPMAS spectra of two ECM samples revealed variations in the polysaccharide content. Analysis of the ECM samples (left) isolated from their corresponding biofilms (right) identified differences in peaks uniquely attributed to polysaccharides. The solid-line spectra are from Fig. 2 and are shown for direct comparison with the dashed-line spectra from a second sample. The percentage of carbon attributed to polysaccharide in the isolated ECM samples ranges from 20 to 43% of the total carbon mass. The error bars represent the standard deviation. Corresponding differences were observed in the intact biofilm. Carbon mass percentages in the ECM were determined by quantitative CPMAS, performing CPMAS as a function of CP time for extrapolation. The intact biofilm analysis is based on the CPMAS spectra shown.

FIG 4

Spectroscopic selection of one-bond and two-bond C-N pairs. ¹³C{¹⁵N}REDOR with a 1.6-ms evolution time identified carbons, and the percentage of those carbons, that are directly bonded to a nitrogen (64,000 scans for both S₀ and S). ¹³C{¹⁵N}REDOR performed with an evolution time of 8.95 ms revealed carbons that were within a two-bond proximity to nitrogen (43,760 scans for both S₀ and S spectra).

FIG 5

Phosphorus contributions to the ECM. (A) ³¹P CPMAS analysis of A. fumigatus ECM. The ³¹P CPMAS spectrum exhibited three overlapping chemical shifts and their associated spinning sidebands, as indicted by ssb (9,168 scans). (B) Spectroscopic selection of carbons near to phosphorous. ¹³C{³¹P}REDOR was performed with an evolution time of 8.95 ms. The REDOR difference spectrum (ΔS, dashed spectrum) reveals the few carbon types that are proximate to ³¹P (38,000 scans for both S₀ and S spectra).