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 native 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. Native hydrated NTM samples were studied at natural abundance without isotope-labelling and any chemical or physical modification. We utilized 1D 13 C and 2D 1 H- 13 C ssNMR spectra and peak deconvolution to identify NTM cell-wall chemical sites. More than ∼100 distinct 13 C signals were identified in the ssNMR spectra. The signals originating from both the flexible and rigid fractions of the native bacteria samples were selectively analyzed by utilizing either CP or INEPT based 13 C 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. We also provide tentative assignments for ∼30 polysaccharides by using well resolved 1 H/ 13 C chemical shifts from the 2D INEPT-based 1 H- 13 C ssNMR spectrum. As an orthogonal way of characterizing the bacteria, electron microscopy (EM) was used to provide spatial characterization. ssNMR and EM data suggest that 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 native NTM strains and identified chemical sites. These results will aid the development of structure-based approaches to combat NTM infections.