Structural insights into native intact Mycobacterium abscessus by conventional and ultrahigh-field solid-state NMR at...

We present an ultrahigh-field magic-angle spinning (MAS) solid-state NMR (ssNMR) study to characterize intact nontuberculous mycobacteria (NTM). Hydrated and dried whole-cell Mycobacterium abscessus samples were investigated by combining conventional high-field ssNMR at 750 MHz with ultrahigh-field ssNMR at 1.2 GHz and ultrafast MAS at 100 kHz. To improve sensitivity and enable multidimensional experiments, ¹³C/¹⁵N isotope labeling was performed after growth in synthetic cystic fibrosis medium (SCFM). We utilized 1D¹³C and 2D ¹H-¹³C and ¹³C-¹³C ssNMR experiments to characterize the chemical composition, dynamics, and structural organization of the M. abscessus cell envelope. The ¹³C isotope-labeling efficiency was found to be non-uniform across different molecular classes, with high incorporation into polysaccharides and lower incorporation into lipid and peptide-associated signals. INEPT- and CP-based experiments selectively probed flexible and rigid fractions of the samples, revealing substantial differences in linewidth, dynamics, and sensitivity between hydrated and dried preparations. Conventional 750 MHz experiments provided high-resolution multidimensional spectra and enabled identification of distinct chemical environments associated with peptidoglycan, arabinogalactan, mycolic acids, lipids, and peptide-associated components. Ultrahigh-field ssNMR at 1.2 GHz combined with ultrafast MAS and ¹H detection improved resolution and sensitivity per mg of sample. The largest improvement of 100 kHz and 1.2 GHz system is observed for the CP spectra of the dry sample, where significantly better FWHM of ¹H: 250 Hz (∼0.2 ppm) and ¹³C: 300 Hz (∼1 ppm) is observed compared to 10 kHz and 750 MHz system with ¹H: 300 Hz (∼0.4 ppm) and ¹³C: 600 Hz (∼3.2 ppm). This allowed the detection of new aromatic and possible nucleic-acid-associated signals. Together, these results demonstrate that ultrahigh-field and ultrafast-MAS ssNMR enables detailed characterization of intact NTM cell envelopes under near-native conditions and provides a framework for future molecular investigations of antimicrobial interactions.