High-resolution 2D solid-state NMR provides insights into nontuberculous mycobacteria

We present a high-resolution magic-angle spinning (MAS) solid-state NMR (ssNMR) study to characterize nontuberculous mycobacteria (NTM). We studied two different NTM strains, Mycobacterium smegmatis, a model, non-pathogenic strain, and Mycobacterium abscessus, an emerging and important human pathogen. Hydrated NTM samples were studied at natural abundance without isotope-labelling, as whole-cells versus cell envelope isolates, and native versus fixed sample preparations. We utilized 1D¹³C and 2D ¹H-¹³C ssNMR spectra and peak deconvolution to identify NTM cell-wall chemical sites. More than ∼100 distinct ¹³C signals were identified in the ssNMR spectra. We provide tentative assignments for ∼30 polysaccharides by using well resolved ¹H/¹³C chemical shifts from the 2D INEPT-based ¹H-¹³C ssNMR spectrum. The signals originating from both the flexible and rigid fractions of the whole-cell bacteria samples were selectively analyzed by utilizing either CP or INEPT based ¹³C ssNMR spectra. CP buildup curves provide insights into the dynamical similarity of the cell-wall components for NTM strains. Signals from peptidoglycan, arabinogalactan and mycolic acid were identified. The majority of the ¹³C signals were not affected by fixation of the whole cell samples. The isolated cell envelope NMR spectrum overlap with the whole-cell spectrum to a large extent, where the latter has more signals. As an orthogonal way of characterizing these bacteria, electron microscopy (EM) was used to provide spatial information. ssNMR and EM data suggest that the M. abscessus cell-wall is composed of a smaller peptidoglycan layer which is more flexible compared to M. smegmatis, which may be related to its higher pathogenicity. Here in this work, we used high-resolution 2D ssNMR first time to characterize NTM strains and identify chemical sites. These results will aid the development of structure-based approaches to combat NTM infections.