Cell wall enrichment unveils proteomic changes in the cell wall during treatment of Mycobacterium smegmatis with sub-lethal concentrations of Rifampicin

In this study, we successfully developed two gel-free approaches to specifically look at cell wall proteins in Mycobacterium smegmatis. The cell wall was subjected to differential centrifugation, differential detergent solubilisation and phase separation to yield the genuine cell wall proteome. The protein extracts were digested by filter-assisted sample preparation for LC-MS/MS analysis on a Q-Exactive mass spectrometer and identified proteins subjected to a strict bioinformatics pipeline. This resulted in the unprecedented coverage of 96 lipoproteins, 475 membrane proteins containing at least one transmembrane helix and 73 secreted proteins. We used this gel-free approach to study the changes in the cell wall proteome during exposure of M. smegmatis to sub-lethal concentration of Rifampicin in a quantitative manner. This enabled us to characterise in detail the dysregulation of transporters such as TatB and ABC transporters, virulence factors such as MCE proteins and PknG, as well as proteins involved in cell wall and lipid synthesis.