Abstract
The lysine acetylation of proteins is a reversible post-translational modification that plays a critical regulatory role in both eukaryotes and prokaryotes. Mycobacterium tuberculosis is a facultative intracellular pathogen and the causative agent of tuberculosis. Increasing evidence shows that lysine acetylation may play an important role in the pathogenesis of M. tuberculosis. However, only a few acetylated proteins of M. tuberculosis are known, presenting a major obstacle to understanding the functional roles of reversible lysine acetylation in this pathogen. We performed a global acetylome analysis of M. tuberculosis H37Ra by combining protein/peptide prefractionation, antibody enrichment, and LC-MS/MS. In total, we identified 226 acetylation sites in 137 proteins of M. tuberculosis H37Ra. The identified acetylated proteins were functionally categorized into an interaction map and shown to be involved in various biological processes. Consistent with previous reports, a large proportion of the acetylation sites were present on proteins involved in glycolysis/gluconeogenesis, the citrate cycle, and fatty acid metabolism. A NAD(+)-dependent deacetylase (MRA_1161) deletion mutant of M. tuberculosis H37Ra was constructed and its characterization showed a different colony morphology, reduced biofilm formation, and increased tolerance of heat stress. Interestingly, lysine acetylation was found, for the first time, to block the immunogenicity of a peptide derived from a known immunogen, HspX, suggesting that lysine acetylation plays a regulatory role in immunogenicity. Our data provide the first global survey of lysine acetylation in M. tuberculosis. The dataset should be an important resource for the functional analysis of lysine acetylation in M. tuberculosis and facilitate the clarification of the entire metabolic networks of this life-threatening pathogen.
Highlights
From the ‡Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; §Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China; ¶Foshan Fourth People’s Hospital, Foshan, China; ʈNational Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
Identification of Lysine-acetylated Proteins in M. tuberculosis H37Ra—Reversible lysine acetylation is emerging as a major regulatory mechanism in metabolism and is an important post-translational modification in both prokaryotes and eukaryotes [7, 49]
To evaluate the diversity and relative abundance of acetylated proteins in M. tuberculosis H37Ra, a ⌬MRA_1161 mutant was constructed and immunoblots of the bacterial cell lysates were probed with an anti-acetyl-lysine antibody
Summary
Construction of the M. tuberculosis H37Ra ⌬MRA_1161 Mutant Strain—Temperature- sensitive specialized transducing plasmids [33] were constructed and used for the allelic-exchange-mediated deletion of the MRA_1161 gene ( designated cobB), which encodes an NADϩ-dependent protein deacetylase in M. tuberculosis H37Ra. All MS/MS samples were analyzed with MASCOT 2.3 (Matrix Science, London, U.K.) and pFind Studio 2.6 [36, 37] to improve the identification of the acetylated peptides Both pFind and MASCOT were used to search a database of forward and reversed M. tuberculosis H37Ra proteins from National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/) that contained 4034 protein sequences. Carbon Source Utilization Experiments—Mycobacterium tuberculosis H37Ra and M. tuberculosis H37Ra DMRA_1161 were grown to mid-log phase in liquid culture medium, collected by centrifugation at 8000 ϫ g for 10 min, and washed three times with 7H9 medium ϩ 0.05% Tween 80 (7H9T) to remove the remaining medium, before they were resuspended in 7H9T and the OD600 adjusted to 0.5. The total numbers of spots were counted and the spot-forming cells in each well was analyzed with a Bioreader 5000 (Dakewei Biotech Company, Shenzhen, China)
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