Characterization of O-mannosylated proteins profiling in bacillus Calmette-Guérin via gel-based and gel-free...

Posttranslational modifications (PTMs) could influence many aspects of protein behavior and function in organisms. Protein glycosylation is one of the major PTMs observed in bacteria, which is crucial for functional regulations of many prokaryotic and eukaryotic organisms. Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been recognized as an indispensable tool in the global fight against tuberculosis (TB) worldwide over several decades. Nevertheless, analysis of glycoprotein profiles of BCG has not been clearly investigated. In this study, we performed O-mannosylated protein analysis in BCG bacteria using gel-based and gel-free approaches. In total, 1,670 hexosylated peptides derived from 754 mannosylated proteins were identified. Furthermore, 20 novel protein products supported by 78 unique peptides not annotated in the BCG database were detected. Additionally, the translational start sites of 384 proteins were confirmed, and 78 proteins were validated through the extension of translational start sites based on N-terminus-derived peptides. The bioinformatic analysis of the O-mannosylated proteins was performed and the expression profiles of four randomly selected proteins were validated through Western blotting. A number of proteins involved in metabolic pathways, including the tricarboxylic acid cycle, glycolysis, oxidative phosphorylation, and two-component system, are discussed. Taken together, these results offer the first O-mannosylated protein analysis of a member of mycobacteria reported to date by using complementary gel-based and gel-free approaches. Some of the proteins identified in this study have important roles involved in metabolic pathways, which could provide insight into the immune molecular mechanisms of this recognized vaccine strain.