Tuberculosis (TB) is one of the leading causes of death worldwide, especially in developing countries. Neonatal BCG vaccination occurs in various regions, but the level of protection varies in different populations. Recently, Mycobacterium vaccae is found to be an immunomodulating therapeutic agent that could confer a significant level of protection against TB. It is the only vaccine in a phase III trial from WHO to assess its efficacy and safety in preventing TB disease in people with latent TB infection. However, the mechanism of immunotherapy of M. vaccae remains poorly understood. In this study, the full genome of M. vaccae was obtained by next-generation sequencing technology, and a proteogenomic approach was successfully applied to further perform genome annotation using high resolution and high accuracy MS data. A total of 3,387 proteins (22,508 unique peptides) were identified, and 581 proteins annotated as hypothetical proteins in the genome database were confirmed. Furthermore, thirty-eight novel protein products not annotated at the genome level were detected and validated. Additionally, the translational start sites of 445 proteins were confirmed, and 98 proteins were validated through extension of their translational start sites based on N-terminus-derived peptides. The physicochemical characteristics of the identified proteins were determined. Thirty-five immunogenic proteins of M. vaccae were identified by immunoproteomic analysis, and twenty of them were selected to be expressed and validated by Western blot for immunoreactivity to serum from patients infected with M. tuberculosis. The results revealed that eight of them showed strong specific reactive signals on the immunoblots. Furthermore，cellular immune response was further examined and one protein displayed a higher cellular immune level in pulmonary TB patients. Twelve identified immunogenic proteins have orthologous in H37Rv and BCG. This is the first study to obtain the full genome and annotation of M. vaccae using a proteogenomic approach, and some immunogenic proteins that were validated by immunoproteomic analysis could contribute to the understanding of the mechanism of M. vaccae immunotherapy.
from #AlexandrosSfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/2uu4mUT