Exploring Mycobacterium Tuberculosis Cell Envelope: Studying Proteins and Lipids Using Styrene Maleic Acid Polymer

Abstract

In 2022, tuberculosis TB caused 1.3 million deaths with nearly 10 million new cases reported, emphasizing the urgent need for new drugs targeting different pathways. The cell envelope of Mycobacterium tuberculosis is an excellent target for novel therapeutics due to its complex architecture, vital for pathogenicity, virulence, and drug resistance. Precise knowledge of molecular interactions on the M. tuberculosis envelope surface is key to identifying crucial components. Current analytical methods involve harsh organic solvents that don't selectively extract outer envelope components. Polymer nano-encapsulation techniques offer promise in studying membrane proteins. Amphipathic polymers like Styrene Maleic Acid (SMA) have been extensively used to directly solubilize proteins and lipids from the cytoplasmic membrane via SMA Lipid Particles (SMALPs). This study presents a protocol for solubilizing and purifying three complexes of an ATP-binding protein ABC transporter from Mycobacterium tuberculosis, predicted to be involved in drug transport as an efflux system. Additionally, this study demonstrates that SMALPs selectively extract peripheral molecules from the cell envelope of mycobacteria. Using [14 C]-uniformly labelled lipids, we show that SMA can extract complex molecules like glycolipids (trehalose monomycolate, trehalose dimycolate, and glucose monomycolate), phospholipids (phosphatidyl inositol mannosides), lipoglycans (lipomannan and lipoarabinomannan), and surface-exposed proteins from the envelopes of Mycobacterium smegmatis without causing cell rupture.