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Κυριακή 18 Φεβρουαρίου 2018

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

Publication date: Available online 18 February 2018
Source:Journal of Proteomics
Author(s): Clemens Hermann, Alexander D. Giddey, Andrew J.M. Nel, Nelson C. Soares, Jonathan M. Blackburn
Understanding the cell wall of mycobacteria is crucial for improving drug design or identifying new antigens suitable to vaccination. Yet this remains problematic due to the complexity of the cell wall composition. In this study, we successfully developed gel-free approaches to study 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. Next, protein extracts were digested by filter-assisted sample preparation for LC-MS/MS analysis on a Q Exactive mass spectrometer, and identified proteins filtered through a stringent bioinformatics pipeline. This yielded the unprecedented coverage of 96 lipoproteins, 475 membrane proteins and 73 secreted proteins. Employing this approach, we next quantified changes in the cell wall proteome during exposure of M. smegmatis to sub-lethal concentration of rifampicin. This facilitated detailed characterisation of the dysregulation of ABC transporters, virulence factors such as Mce proteins and PknG, and proteins involved in cell wall and lipid synthesis. Crucially, these cell wall proteins are under-represented in previous proteome analysis of M. smegmatis. This approach enables further quantitative proteomic studies of the role of the cell wall proteome of mycobacteria in virulence or during drug exposure.SignificanceWe developed novel gel-free sample preparation workflows for the cell wall fraction of mycobacteria that significantly increase the coverage of the cell wall proteome compared to previous studies. We then provide a data analysis workflow that enables the removal of likely cytosolic contaminants in the cell wall fraction post-measurement. Combined, these approaches increase the coverage of the cell wall proteome while ensuring that the identified proteins are true cell wall proteins and not carry-over of high-abundance contaminants from the cytosol. We have applied these approaches to quantify the dysregulation of cell wall proteins during exposure of M. smegmatis to rifampicin, which has shed new light on the coordinated down-regulation of ABC transporters as well as virulence factors present in the cell wall proteome.

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