Proteome-wide lysine acetylation profiling of the human pathogen Mycobacterium tuberculosis.

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Proteome-wide lysine acetylation profiling of the human pathogen Mycobacterium tuberculosis

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Longxiang Xie a , Xiaobo Wang b , Jie Zeng a , Mingliang Zhou a , Xiangke Duan a , Qiming Li a , Zhen Zhang a , Hongping Luo a , Lei Pang a , Wu Li a , Guojian Liao a , Xia Yu b , Yunxu Li b , Hairong Huang b,∗∗ , Jianping Xie a,∗ a Institute of Modern Biopharmaceuticals, State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three Gorges Area, Key Laboratory of Eco-environments in Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Southwest University, Beibei, Chongqing 400715, China b National Clinical Laboratory on Tuberculosis, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China

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Article history: Received 14 June 2014 Received in revised form 5 October 2014 Accepted 21 November 2014 Available online xxx

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Keywords: Nε -lysine acetylation PTM 21 Mycobacterium 22 Virulence 23 Persistence 24 Q2 Antibiotic resistance 25 19 20

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Nε -Acetylation of lysine residues represents a pivotal post-translational modification used by both eukaryotes and prokaryotes to modulate diverse biological processes. Mycobacterium tuberculosis is the causative agent of tuberculosis, one of the most formidable public health threats. Many aspects of the biology of M. tuberculosis remain elusive, in particular the extent and function of Nε -lysine acetylation. With a combination of anti-acetyllysine antibody-based immunoaffinity enrichment with high-resolution mass spectrometry, we identified 1128 acetylation sites on 658 acetylated M. tuberculosis proteins. GO analysis of the acetylome showed that acetylated proteins are involved in the regulation of diverse cellular processes including metabolism and protein synthesis. Six types of acetylated peptide sequence motif were revealed from the acetylome. 20 lysine-acetylated proteins showed homology with acetylated proteins previously identified from Escherichia coli, Salmonella enterica, Bacillus subtilis and Streptomyces roseosporus, with several acetylation sites highly conserved among four or five bacteria, suggesting that acetylated proteins are more conserved. Notably, several proteins including isocitrate lyase involved in persistence, virulence and antibiotic resistance are acetylated, and site-directed mutagenesis of isocitrate lyase acetylation site to glutamine led to a decrease of the enzyme activity, indicating major roles of KAc in these proteins engaged cellular processes. Our data firstly provides a global survey of M. tuberculosis acetylation, and implicates extensive regulatory role of acetylation in this pathogen. This may serve as an important basis to address the roles of lysine acetylation in M. tuberculosis metabolism, persistence and virulence. © 2014 Published by Elsevier Ltd.

1. Introduction Post-translational modifications are important for eukaryotic and prokaryotic protein function. Protein lysine acetylation (normally referred to Nε -lysine acetylation), a dynamic and reversible PTM, is the transfer of acetyl moiety from acetyl-CoA to the ␧amino groups of lysine residues in proteins (Hu et al., 2010; Xie et al., 2012). Nε -lysine acetylation not only can alter DNA binding activity and thus gene expression, but also regulate protein–protein

∗ Corresponding author. Tel.: +86 2368367108; fax: +86 2368367108. ∗∗ Corresponding author. Tel.: +86 10 89509159; fax: +86 10 89509159. E-mail addresses: [email protected] (H. Huang), [email protected] (J. Xie).

interactions, protein activity, mRNA stability, coordinating carbon source utilization, and metabolic flux (Hu et al., 2010; Wang et al., 2010; Zhao et al., 2010). Since the discovery of lysine acetylation in almost fifty years ago, the studies of this protein modification have focused primarily on histone proteins and other transcriptionassociated proteins in nucleus (Phillips, 1963; Allfrey et al., 1964). However, subsequent studies found the widespread of this modification in non-histone proteins in eukaryotes (human, mouse, plant, drosophila and protozoan) and these acetylated non-histone proteins involved in various cellular physiology including central metabolism, protein synthesis and cell cycle, which indicates that lysine acetylation extends far beyond transcription (Weinert et al., 2011; Choudhary et al., 2009; Finkemeier et al., 2011; Jeffers and Sullivan, 2012; Kim et al., 2006). As many acetylated proteins were identified in the mitochondrion, a close relative of

http://dx.doi.org/10.1016/j.biocel.2014.11.010 1357-2725/© 2014 Published by Elsevier Ltd.

Please cite this article in press as: Xie L, et al. Proteome-wide lysine acetylation profiling of the human pathogen Mycobacterium tuberculosis. Int J Biochem Cell Biol (2014), http://dx.doi.org/10.1016/j.biocel.2014.11.010

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␣-proteobacteria, it is very likely that massive protein lysine acetylation might also occur in bacteria, not restrict to eukaryotic cells. The first bacterial lysine acetylome was studied in one member of the ␥-proteobacteria phylum, Escherichia coli, and 144 acetylated proteins were identified (Yu et al., 2008; Zhang et al., 2009a). To date, the global lysine acetylation profiles of other prokaryotes such as E. coli, S. enterica, B. subtilis, thermophilic Geobacillus kaustophilus, Erwinia amylovora Vibrio parahaemolyticus and S. roseosporus have also been intensively explored and most acetylated proteins are involved in nearly all cellular processes, such as central metabolism, protein translation and virulence (Wang et al., 2010; Wu et al., 2013; Kim et al., 2013; Lee et al., 2013; Zhang et al., 2013; Pan et al., 2014; Liao et al., 2014a). These studies implicated that protein lysine acetylation is widespread and important PTM which might rival with phosphorylation in bacteria (Kouzarides, 2000). Tuberculosis remains a formidable infectious disease with onethird of the world population latently infected by the causative agent Mycobacterium tuberculosis, 9 million new TB cases each year, and 1.6 million deaths worldwide. TB is the leading cause of death in people coinfected with HIV. This is exacerbated by the emergence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) (Shin et al., 2004). USP, a universal stress protein, was the first characterized acetylated protein in mycobacterium that can be acetylated by both M. tuberculosis cAMP-regulated protein lysine acetyltransferase Rv0998 and its Mycobacterium smegmatis ortholog MSMEG 5458 (Nambi and Basu, 2010). Subsequently, acetyl-CoA synthetase (Szabo et al., 2014), one acetate catabolic enzyme in M. tuberculosis, was demonstrated to be acetylated by purified M. smegmatis lysine acetylase (MSMEG 5458) in vitro. Recently, multiple FadD enzymes involved in the metabolism of fatty acids were found to be acetylated by Rv0998 in a cAMPdependent manner (Nambi et al., 2013). A specific fatty acyl-AMP ligase FadD33 (also known as MbtM) required for mycobacterial siderophore synthesis was also acetylated by mycobacterium protein lysine acetyltransferase in the same manner and this acetylation would lead to enzyme inhibition (Vergnolle et al., 2013). Given the widespread regulatory role of lysine acetylation, it is tempting to speculate that M. tuberculosis proteome has more as yet unidentified acetylated proteins. To explore the abundance and biological significance of lysine acetylation in M. tuberculosis, proteomics is used in combination with immunoprecipitation to identify the acetylated protein. Here we report the systematic identification of the lysine acetylome of M. tuberculosis, namely 1128 unique acetylation sites in 658 acetylated proteins with various biological functions and cellular location. The lysine-acetylated proteins are mainly involved in central metabolism and protein synthesis. Interestingly, several proteins involved in M. tuberculosis persistence, virulence and antibiotic resistance were also found to be lysine acetylated. These results provide a global picture of the acetylome of M. tuberculosis.

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2. Materials and methods

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2.1. Strain culture and protein extraction

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M. tuberculosis H37Rv was grown to mid-log phase in 7H9 media with 0.05% Tween 80, albumin, dextrose, and catalase supplement (Cole et al., 1998). The collected sterilized strains were then stored as 1-mL aliquots in 15% glycerol (final concentration) at 80 ◦ C. For individual experiments, bacilli were grown in roller bottles in Middlebrook 7H9 medium with 0.05% Tween 80 and ADC supplement at 37 ◦ C. The cultured cells were harvested and washed twice with cold phosphate-buffered saline (PBS), then lysed in 8 M urea supplemented with 1 mM DTT, 2 mM EDTA, protease inhibitor cocktail (Protease Inhibitor Cocktail Set III; Calbiochem), and HDAC

inhibitor (30 mM nicotinamide, 50 mM sodium butyrate, 3 ␮M Trichostatin A). This was then sonicated with 12 short bursts of 10 s intervals followed by 30 s intervals for cooling. Unbroken cells and debris were removed by centrifugation at 4 ◦ C for 10 min at 20,000 × g. Protein content in supernatant was defined with 2-D Quant kit (GE Healthcare) according to the manufacturer’s instructions and precipitated with 20% trichloroacetic acid overnight at 4 ◦ C. The resulting precipitate was then washed three times with ice-cold acetone. The air-dried precipitate was resuspended in 100 mM NH4 HCO3 and then digested with trypsin (Promega) at an enzyme-to-substrate ratio (1:50) at 37 ◦ C for 12 h. The tryptic peptides were reduced with 5 mM dithiothreitol for 45 min at 56 ◦ C and then alkylated with 15 mM iodoacetamide at room temperature for 30 min in complete darkness. The reaction was finally terminated with 15 mM cysteine for 30 min at room temperature. To ensure complete digestion, additional trypsin at an enzyme-to-substrate ratio (1:100) was added, and the mixture was incubated for an additional 4 h. The digested peptides were freeze-drying in a SpeedVac (Thermo Scientific). 2.2. Enrichment of lysine acetylated peptides The trypic digest was redissolved in NETN buffer (50 mM Tris, 1 mM EDTA, 100 mM NaCl, 0.5% Nonidet P-40, pH 8.0) and incubated with anti-acetyllysine agarose beads (PTM Biolabs) at 4 ◦ C overnight with a gentle oscillation. After incubation, the beads were carefully washed three times with NETN buffer, twice with ETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris, pH 8.0) and once with water. The bound peptides were eluted from the beads by 1% trifluoroacetic acid and dried in the SpeedVac. Prior to HPLC/MS/MS analysis, the obtained peptides were rinsed with C18 ZipTips (Millipore) according to the manufacturer’s instructions. 2.3. LC-ESI–MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry) analysis by Q exactive The peptides were resuspended in buffer A (0.1% FA, 2% ACN) and centrifuged at 20,000 × g for 2 min. The supernatant was transferred into a sample tube and loaded onto an Acclaim PepMap 100 C18 trap column (Dionex, 75 ␮m × 2 cm) by EASY nLC1000 nanoUPLC (Thermo Scientific) and the peptide was eluted onto an Acclaim PepMap RSLC C18 analytical column (Dionex, 50 ␮m × 15 cm). A 34 min gradient was run at 300 nL/min, starting from 5% to 30% B (80% ACN, 0.1% FA), followed by 2 min linear gradient to 40% B, 2 min to 80% B, and maintenance at 80% B for 4 min. The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q Exactive (Thermo Scientific) coupled online to the UPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using 25% NCE with 4% stepped NCE. Ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 15 MS/MS scans was applied for the top 15 precursor ions above a threshold ion count of 4E4 in the MS survey scan with 2.5 s dynamic exclusion. The electrospray voltage applied was 1.8 kV. Automatic gain control was used to prevent overfilling of the ion trap; 2E5 ions were accumulated for generation of MS/MS spectra. For MS scans, the m/z scan range was 350–1800 Da. 2.4. Data processing The identification of protein and acetylation site was performed by MaxQuant with integrated Andromeda search engine (v. 1.3.0.5). Tandem mass spectra were searched against Uniprot M. tuberculosis H37Rv protein database (4222 sequences, 2009) concatenated with reverse decoy database and protein sequences of common


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contaminants. Trypsin/P was specified as cleavage enzyme allowing up to 3 missing cleavages, 4 modifications per peptide and 5 charges. Mass error was set to 6 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on Cys was specified as fixed modification and oxidation on Met, acetylation on Lys and acetylation on protein N-terminal were specified as variable modifications. False discovery rate (FDR) thresholds for protein, peptide and modification site were specified at 0.01. Minimum peptide length was set at 7. All other parameters in MaxQuant were set to default values. Lys acetylation site identifications with localization probability less than 0.75 or from reverse or contaminant protein sequences were removed. 2.5. Bioinformatics analysis Protein functional annotation Gene Ontology (GO) annotation proteome was performed using the UniProt-GOA Database (http://www.ebi.ac.uk/GOA/). Proteins were categorized into biological process, cellular compartment and molecular function according to Gene Ontology annotation. Kyoto Encyclopedia of Genes and Genomes (KEGG) were utilized to annotate pathways: firstly, using KEGG online service tools KAAS to annotate proteins, secondly, using KEGG online service tools KEGG mapper to map on the KEGG pathway database, finally, using InterPro database and InterProScan to annotate protein domains and applying CORUM database to annotate protein complex. Functional enrichment analysis We used the Fisher’s exact test to check the enrichment or depletion (two-tailed test) of specific annotation terms among members of resulting protein clusters. Then through the method proposed by Benjamini and Hochberg, we further adjust the derived p-values to address multiple hypotheses. Any terms with adjusted p-values below 0.05 in any of the clusters were treated as significant. Acetylated protein secondary structure prediction The local secondary structures were carried out according to NetSurfP software. The different secondary structure probabilities of identified acetylated residues in this study were compared with the secondary structure probabilities at the position of control residues containing all Lys residues in our database. The distribution of acetylated and non-acetylated amino acids in protein secondary structures was analyzed. Model of sequences around acetylation site analysis The analysis of enrichment or depletion of amino acids at specific positions of acety-13-mers (6 amino acids upstream and downstream of acetylation sites) in all protein sequences was performed by software motif-X. The background database parameter was all protein sequences in the database. 2.6. Cloning and mutagenesis of Rv0467 protein The list of primers used for PCR and mutagenesis are provided in Table S1. The Rv0467 gene was amplified by using primers Rv0467A and Rv0467D, with the genomic DNA of M. tuberculosis H37Rv as the template. The product was digested with BamH I and Hind Ш and cloned into similarly digested pET28 to generate pET28Rv0467. The clone was confirmed by sequencing (BGI, China). Point mutations in Rv0647 were generated by site-directed mutagenesis. Using primers Rv0467K322Q and Rv0467K322R, the mutation genes were obtained through overlap extension PCR. The mutation gene was cloned into pET28 for sequencing.

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Supplementary Table S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.biocel.2014.11.010. 2.7. Expression and purification of His-tagged proteins Both wide type and mutant proteins were expressed in the E. coli BL21 on induction using 0.025 mmol/L isopropyl-␤-thio-galactopyranoside. Cells were suspended in buffer 1 (pH 8.0) containing 50 mM NaH2 PO4 and 300 mM NaCl with protease inhibitors, and then were sonicated the suspension on ice for 40 min. Cell debris and membranes were removed by centrifugation at 12,000 rpm/min. The supernatant was incubated with Ni2+ -nitrilotriacetate column. After sample application, the column was washed with buffer 2 (same as buffer 1 but with 20 mM imidazole, pH 8.0), followed by a wash with buffer 3 (same as buffer 1 but with 50 mM imidazole, pH 8.0). The bound proteins were eluted from the column with buffer 4 (same as buffer 1 plus 10% glycerol, 300 mM imidazole, pH 8.0). Fractions containing isocitrate lyase activity were pooled and frozen at −70 ◦ C until further use. 2.8. Enzyme activity assay (Honer Zu Bentrup et al., 1999) By using 96-well plates, the right amount of MOPS buffer (50 mM MOPS, 5 mM MgCl2 , 5 mM l-cysteine and 1 mM EDTA, pH 6.8) was firstly mixed with 2 U lactate dehydrogenase (LDH), 40 ␮L 2 mM NADH at 37 ◦ C for 20 min. Then, adding 100 ␮L 20 mM isocitric acid trisodium salt to monitor the reduction of NADPH at 340 nm with a Thermo Scientific Microplate (Spectra Max 1970).

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3. Results and discussion

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3.1. Profiling of M. tuberculosis H37Rv lysine acetylome

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To identify the M. tuberculosis acetylome in vivo, proteins were isolated from exponentially growing cells. Proteins were digested with trypsin to yield tryptic peptides and acetylated peptides were enriched with specific acetyllysine antibody. The enriched acetylated peptides were analyzed by LC-ESI–MS/MS. The obtained MS/MS spectra were used to search against the Uniprot M. tuberculosis H37Rv databases. We identified 1128 acetylation peptides with a peptide score greater than thirty (Table S2). These peptides, which exhibit different abundances depending on their length (Fig. 1A), match 658 acetylated proteins. We compared the M. tuberculosis acetylome to previously reported bacterial acetylomes. The result is presented in Table 1. The identified acetyl proteins account for 16.37% of the total proteins in the M. tuberculosis, which is the highest ratio of acetyl proteins that has so far been identified in bacteria. This reflects the extensive acetylation in M. tuberculosis. Supplementary Table S2 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.biocel.2014.11.010. Previously the detection of protein lysine acetylation was challenging due to the requirement of relatively massive material and the difficulties of analysis in distinguishing this modification from tri-methylation. With the availability of more sensitive analytical instrumentation and better reagents, most problems have been solved (Kim et al., 2006). For example, the availability of tandem MS has allowed the identification of acetylated peptides unambiguously, and the development of high-quality anti-acetyllysine antibody has made the protocol for the affinity enrichment of lysine-acetylated peptides more effective. The percentages of E. coli acetyl proteins from three studies (2.1%, 2.2%, and 8.2%) performed in different years (2008, 2009 and 2013) confirmed this (Yu et al., 2008; Zhang et al., 2009b, 2013). Now many groups


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Fig. 1. (A) Distribution of acetylated peptides based on their length. (B) Distribution of acetylated proteins based on their number of acetylation peptides.

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including our group utilize the following strategy to investigate the proteome-wide identification of lysine acetylation: protein fractions from cells were firstly digested with trypsin, then the acetylated peptides were subjected to immune-affinity purification with an antibody specific for acetyl-lysine and finally the isolated peptides were analyzed by LC/MS/MS for peptide identification (Wu et al., 2013; Kim et al., 2013; Lee et al., 2013; Zhang et al., 2013). However, if assessing the degree of particular acetylated peptides under different growth conditions, stable isotope labeling by amino acids in cell culture (SILA need be used for quantitative

comparisons. For example, to identify and quantify changes in bacterial lysine acetylation in response to glucose, Gozde et al. prepared the experimental and control cell samples in such media supplemented with the isotopically labeled heavy amino acid or with a light form of that amino acid (Colak et al., 2013). Following the preparation of tryptically digested peptides from cells, samples were equally combined and fractionated via ion exchange HPLC. Using agarose beads conjugated with pan-anti-succinyllysine antibody, acetylated peptides were enriched and then analyzed via nano-HPLC-MS/MS to identify acetylated peptides and quantify the

Table 1 Comparison of M. tuberculosis H37Rv acetylome with other published bacteria acetylome. Organism

Strains

Genome size (Mb)

Proteins

No. of AcK-proteins

No. of AcK-sites

% of acetylation

Reference

Mycobacterium tuberculosis Streptomcyes roseosporus Bacillus subtilis Escherichia coli Escherichia coli Escherichia coli Salmonella enterica Thermus thermophilus Geobacillus kaustophilus Erwinia amylovora

H37Rv NRRL15998 168 W3110 DH5 DH10 Serovar typhimurium LT2 HB8 HTA426 Ea1189 Ea273

4.41 7.81 4.22 4.64 4.64 4.64 4.95 2.12 3.54 3.91

4222 6924 4175 4144 4144 4141 4556 2173 3539 3565

658 667 185 85 91 349 191 128 114 96

1128 1143 332 125 138 1070 260 197 253 141

16.37% 9.68% 4.48% 2.05% 2.20% 8.42% 4.22% 5.72% 7.15% 2.7%

This study Liao Dooil Kim Zhang Junmei Zhang Kai Zhang Wang Okanishi Dong-Woo Lee Xia Wu


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relative abundant (Colak et al., 2013). Now, several factors might compromise the exhaustiveness of global analysis of acetylated peptides, such as sample complexity and the fact that abundant acetylated peptides maintain their dominant position in analyses. With improved methodology, these limiting factors might be overcome.

3.1.1. Distribution of acetylation sites in M. tuberculosis H37Rv proteome To evaluate the distribution of acetylation sites in M. tuberculosis lysine acetylome, we calculated the modification sites number of every acetylated protein. 223 proteins (34%) in our dataset were acetylated on multiple lysine (Fig. 1B), including 9 on Rv0951; 13 on Rv0350; 14 on Rv0668. GroEL2 contained the highest number of lysine acetylated sites (24) reported to date. Why some proteins have multiple acetylation sites and whether multiple acetylations occur simultaneously or stepwise remain to be explored. In particular, among the 771 essential M. tuberculosis genes previously defined by Griffin et al. (Zhang et al., 2012), 202 (26.2%) proteins were found to be acetylated, consistent with that 51 (18.8%) proteins were found to be acetylated among the 271 essential B. subtilis genes, implicating a physiological role of lysine acetylation in M. tuberculosis.

3.1.2. Cellular localization and functional annotations of acetylated proteins To further characterize the lysine acetylome in M. tuberculosis, we performed gene ontology (GO) functional classification of all acetylated proteins based on biological process and molecular function (Fig. 2). The classification results for biological process and molecular function both showed that the largest group of acetylated proteins consists of enzymic proteins associated with metabolism, which accounts for 38% and 51% of the whole acetyl proteins, respectively (Fig. 2A and B). These findings are consistent with previous observations that most lysine acetylated proteins are involved in metabolism. The second largest group in term of their molecular function is the set of binding proteins, and the number of proteins in this group is 39% of all identified proteins. Another large acetyl protein group determined by biological process is associated with cellular process, which account for 33% of all identified proteins. The GO analysis indicates that acetylated proteins have diverse biological process and molecular functions in M. tuberculosis. The subcellular location of the acetyl proteins was also analyzed, and the results showed that 537 of the acetylated proteins are frequently occurred in the cytoplasm (82%), 30 proteins are distributed in the extracellular (4%), 91 proteins are located in membrane (14%), as shown in Fig. 2C. This overall trend in the sub-cellular location of the acetylated proteins is consistent with our previous observation in S. roseosporus (Liao et al., 2014a). Furthermore, to reveal the preferential types of proteins targets for lysine acetylation, we perform GO enrichment analysis for the acetylation data. In the biological process category, the process related with translation, tRNA aminoacylation, amino acid activation and cellular protein metabolic process were found to be significantly enriched (Fig. 3A and Table S3). The enrichment result determined through the molecular function that protein with aminoacyl-tRNA ligase activity also has a higher tendency to be acetylated (Fig. 3A and Table S3). Moreover, KEGG pathway analysis showed that proteins involved in ribosome and aminoacyl-tRNA biosynthesis were highly enriched (Fig. 3B and Table S3). Overall, these data indicate that lysine acetylation might play a regulatory role in protein biosynthesis.

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Supplementary Table S3 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.biocel.2014.11.010. 3.2. Patterns of acetylated peptides 3.2.1. Local structural properties of acetylated lysines Acetylated lysine was previously shown to prefer specific protein secondary structures in comparison with non-acetylated lysine. Most identified KAc sites from E. amylovora and E. coli were found in loops or helices (Wu et al., 2013), similar to the findings from human lysine acetylome (Lu et al., 2011). To find whether this correlation holds true for the M. tuberculosis lysine acetylome, we used NetSurfP software for lysine acetylation analysis and found that approximate 28% (316) of the acetylation sites were located at regions of ordered secondary structure. Among them, 248 were located in ␣-helix and 68 sites were in a ␤-sheet. The remained 72% (812) acetylated sites located in unstructural regions of the protein. All lysines counted, about 71% are predicted to be in disordered regions (Fig. 4A). This argues against the notion that acetylation sites have local structural preferences as seen in E. amylovora, E. coli and eukaryote. The secondary structure preferences for acetylation sites might vary with species. 3.2.2. Lysine acetylation motifs Conserved protein sequence motifs are related with several post-translational modifications such as protein phosphorylation. To test whether linear lysine acetylation motifs exist among lysine-acetylated proteins, we analyze the global acetylated substrates using motif-x software. Six conserved motifs can be found among these acetylated peptides, namely Q1 (Kac ****R), Q2 (Kac H), Q3 (Kac Y), Q4 (Kac ****K), Q5 (Kac ***K), Q6 (Kac ***R) and exhibit distinct abundances (Fig. 4B and C). Enrichment of a positively charged residue including lysine (K), arginine (R) or histidine (Y), and a residue with aromatic group such as tyrosine (Y) toward the C terminus was common among these motifs, suggesting the lysine acetyltransferases might prefer the polypeptide with two distinct types of residues as substrate. Lysine acetyltransferase Rv0998 can acetylate FadD33 and ACS at K511, K617 respectively, with surrounding residues belonging to the Q6 motif (Vergnolle et al., 2013). It is tempting to test whether other acetylated proteins bearing this motif are the substrates of Rv0998. Intriguingly, two acetylated lysine motifs (K ac H and K ac Y) are observed in human cells and E. coli (Zhang et al., 2009b; Kim et al., 2006). All six acetylated lysine motifs are present in the S. roseosporus. These results indicate that lysine acetyltransferases are conserved and widespread. 3.3. Similarities and differences between M. tuberculosis acetylome and other bacterial acetylomes To find the feature of KAc among diverse organisms, the lysine acetylome of M. tuberculosis was compared with known KAc proteins from E. coli, S. enterica, B. subtilis and S. roseosporus (Wang et al., 2010; Kim et al., 2013; Zhang et al., 2013; Liao et al., 2014b). Fig. 5A showed that among the 658 acetylated proteins, 93, 65, 46 and 240 proteins have orthologs in the acetylome of E. coli, S. enterica, B. subtilis and S. roseosporus, respectively. It was not surprise to find that the M. tuberculosis acetylome profile exhibited greater similarity with gram-positive bacteria S. roseosporus, due to their closer phylogeny and acetylation abundance. 20 acetylated proteins were shared among the five strains (Table S4): 14 proteins involved in cellular metabolism, 5 proteins related with protein synthesis (Elongation factor G, 50S ribosomal protein L2, L4, translation initiation factor IF-2), one superoxide dismutase, implying conserved and important function among bacteria. We also aligned M. tuberculosis acetylated peptides with those from E. coli,


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Fig. 2. Gene ontology functional classification of the identified acetylation proteins based on biological processes (A); molecular function (B); subcellular location (C).

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S. enterica, B. subtilis, S. roseosporus and found several acetylated peptides conserved between two, three or four bacteria, for example, one lysine acetylation site of inorganic pyrophosphatase conserved in E. coli, S. enterica, S. roseosporus and M. tuberculosis, but another lysine acetylation site is conserved in E. coli, S. roseosporus and M. tuberculosis (Table S4). These results indicated that the acetylation level of same acetylated protein varies with species. Supplementary Table S4 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.biocel.2014.11.010.

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3.4. Proteins can be acetylated, phosphorylated and pupylated

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Acetylation and other PTMs form complex regulatory networks, and this “crosstalk” largely remains an unexplored interesting topic in pathogen. Using a mass spectrometry-based approach, Prisic

et al. identified 301 phosphoproteins and 500 phosphorylation sites in M. tuberculosis proteins (Prisic et al., 2010). The size of phosphoproteome was far smaller than acetylproteome (658) found in this study. In addition, 61 proteins from M. tuberculosis were shared between lysine-acetylation and S/T/Y-phosphorylation (Fig. 5B and Table S5). Pupylation is a post-translational protein-to-protein modification system in prokaryotes and prokaryotic ubiquitin-like protein targets several proteins for degradation by a bacterial proteasome in a manner similar to ubiquitin (Ub) mediated proteolysis in eukaryotes (Guzman et al., 2004). We found that 236 proteins accounting for 39% (236/604) of total pupylation proteins reported by Richard et al. were acetylated, most of which are involved in intermediary metabolism, protein translation (Fig. 5B and Table S6). Above all, among acetylation and phosphorylation sharing proteins, 17 identified lysine acetylation sites were found adjacent to (S/T/Y) phosphorylation sites (Table S5). Furthermore, among 10


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Fig. 3. Enrichment analysis of the acetylated proteins in M. tuberculosis. (A) The acetylated proteins in each group are significantly overrepresented; as classified by their GO

Q5 annotation in terms of biological process (blue bars) and molecular function (red bars). (B) KEGG pathway enrichment analysis. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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acetylation and pupylation co-existing proteins, 13 lysine residues were found to localize in the same position with pupylation (Table S6). As well, the acetylation of heat shock protein HspX was identified at four lysine sites (K64, K85, K114 and K132), where pupylation was reported. It is potentially possible that lysine

acetylation affects the function of phosphorylation nearby or competes with other lysine PTMs at the same lysine residues. In phosphoproteomic study, M. tuberculosis was cultured under several conditions (oxidative stress, NO stress, hypoxia, and glucose or acetate as a carbon source) and harvested at different growth


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Fig. 4. Statistics of lysine acetylation sites. (A) Distribution of secondary structures containing lysine acetylation sites. Different secondary structures (␣ helix, ␤ strand and coil) of acetylated lysine identified in this study were compared with the secondary structure of all lysine on all proteins. (B) Acetylation motifs and conservation of acetylation sites. The enrichment of amino acids in specific positions of acetylated lysine-13-mers (6 amino acids upstream and downstream of the acetylation site) is used by Motif-X software. (C) The number of identified peptides containing acetylated lysine in each motif.

Fig. 5. (A) Comparison of M. tuberculosis acetylproteome with those of E. coli, B. subtilis and S. enterica and S. roseosporus. Common lysine-acetylated proteins from the datasets of E. coli, S. enterica, B. subtilis, S. roseosporus and M. tuberculosis were listed in the table. (B) The Venn diagram illustrating the lysine-acetylated, the S/T/Y-phosphorylated and the pupylated M. tuberculosis proteins.

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stages (log phase and stationary phase) to be analyzed (Prisic et al., 2010). However, in pupylome research and our acetylome study, M. tuberculosis was just grown in Middlebrook 7H9 broth under normal condition and collected at log phase (OD580 ≈ 1.0. Among these lysine-acetylation and phosphorylation common proteins, 36 proteins were phosphorylated at log phase and most of them were involved in metabolism. In particular, M. tuberculosis encoding one Ser/Thr protein kinase (STPK), PknG, can phosphorylate the forkhead-associated domain-containing protein GarA to regulate enzymes of central carbon and nitrogen metabolism (Cowley et al., 2004). This STPK and GarA can be both acetylated and phosphorylated at log phase, indicating two post-translational modifications might coexist on these proteins at the same time and regulate the metabolic process together. The contribution ratio of the two PTMs to the regulation remains to be determined. Supplementary Tables S5 and S6 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.biocel.2014.11.010.

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3.5. Lysine acetylated proteins are involved in central metabolism

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Several proteins involved in fatty acid and propionate metabolism in M. tuberculosis have been previously reported (Nambi et al., 2013). As expected, nearly all enzymes involved in central metabolism including glycolysis and gluconeogenesis, pentose phosphate pathway (PPP) and citrate cycle are also

acetylated (Fig. S1). Isocitrate lyase (ICL1, Rv0467), the key enzyme of M. tuberculosis glyoxylate cycle, was identified to be acetylated at K322 (Fig. 6A). S. enterica isocitrate lyase (AceA) could be acetylated at K34 in vitro, and the increase of AceA acetylation led to 30% loss in AceA-specific activity (Wang et al., 2010). Therefore, it is interesting to study the effect of K322 acetylation on the function of ICL1. We firstly carried out sequence alignment and studied possible roles of the identified acetylated lysine residues based on the protein structures. We found that acetylated lysine residue (K322) is not evolutionarily conserved among mycobacteria and is not directly involved in substrate binding and catalysis (Fig. 6B). To explore the possible function of lysine acetylation, we carried out mutagenesis experiment. The modified lysine residue K322 were mutated to either arginine (K-to-R mutant), which retained the positive charge, or glutamine (K-to-Q mutant), which mimicked lysine acetylation. Each mutant protein were expressed and then purified up to >90% purity (Fig. 6C). The measured enzymatic activities of the mutant proteins were: WT, 15.33U; K322Q, 0.526U; K322R, 25.16U. In comparison with enzyme activity of wide type ICL protein, the mutation of K322 to glutamine led to a decrease of the enzyme activity, while K-to-R mutant also has enzyme activity (Fig. 6D), indicating that K322 is critical for the activity of isocitrate lyase and that lysine acetylation is likely to inhibit its enzymatic function in M. tuberculosis. Supplementary Fig. S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.biocel.2014.11.010.


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Fig. 6. MS/MS spectra of acetylpeptides and mutagenesis analysis of isocitrate lyase. (A) Acetyl-peptide K(ac)HLDDATIAK with acetylation site at K322. (B) DNAMAN alignment of isocitrate lyase homologs from M. abscessus (GenInfo Identifier (GI): 491203798), M. marinum (GI: 183174142), M. avium (GI: 665989294), M. gilvum (GI: 145213198), M. bovis (GI: 61217913), M. smegmatis (GI: 118471509) and M. tuberculosis. Conserved sites are shaded with purple. Acetyl-lysine residue is indicated by red triangles, respectively. The positions are labeled corresponding to the M. tuberculosis sequence. (C) Purity of wide type and mutated proteins shown by SDS-PAGE gel. (D) Enzymatic activities of WT, K322Q and K322R isocitrate lyase. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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3.6. Protein related with persistence, virulence and antibiotic resistance are acetylated As one of most successful pathogens of human being, M. tuberculosis owns three “magic weapons”, asymptomatic persistence, virulence and antibiotic resistance. 16 acetylated proteins including isocitrate lyase are reported to be involved in M. tuberculosis persistence (Honer zu Bentrup and Russell, 2001; Stewart et al., 2003; Shi and Zhang, 2010) (Table S7). Already mentioned in the above, acetylation of K322 in ICL could lead to the significant decrease of enzyme activity. Previous study had demonstrated that disruption of the ICL gene essential for the metabolism of fatty acids in M. tuberculosis attenuated bacterial persistence and virulence in immune-competent mice, but had no significant effect on acute replication (McKinney et al., 2000; Munoz-Elias and McKinney, 2005). Furthermore, ICL could mediate broad antibiotic tolerance in M. tuberculosis (Nandakumar et al., 2014). ICL-deficient Mtb strains were significantly more susceptible than wild-type Mtb to three clinically used tuberculosis drugs, isoniazid (Pinheiro et al., 2014), rifampicin (RIF) and streptomycin (Nandakumar et al., 2014). It is tempting to suggest that acetylation of ICL plays a role in M. tuberculosis persistence, virulence and antibiotic resistance. Supplementary Table S7 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.biocel.2014.11.010. Apart from ICL, there are 19 acetylated proteins related with virulence and 9 acetylated proteins involved in antibiotic resistance (Table S7). For example, secretory apparatus esx-1 locus is highly conserved and widespread among Mycobacteria, which has been intensely studied due to its association with M. tuberculosis virulence (Coros et al., 2008). Five proteins encoded by the esx-1 locus, namely Rv3868 (EccA1), Rv3870 (eccCa1),

Rv3871 (Eccb1), Rv3882c (EccE1), were acetylated (Fig. S2). InhA, an NADH-dependent enoyl-ACP reductase potently inhibited by INH-NAD adduct (Dessen et al., 1995; Vilcheze and Jacobs, 2007), is found to be identified at K165. The acetylation residue of inhA (K165) was the cofactor binding site, and replacement of K165 with alanine (A) and methionine (M) were unable to bind NADH (Parikh et al., 1999). Therefore, it will be interesting to determine the effect of acetylation of key lysine on the function of these proteins implicated in virulence and antibiotic resistance.

4. Conclusion In brief, lysine acetylation has recently been demonstrated a dynamic, reversible, and regulatory post-translational modification in bacteria. We found 1028 acetylation sites on 658 proteins involved in a broad spectrum of fundamental cellular processes ranging from regulation of metabolic pathways to protein synthesis. The data confirmed the generality of N␧ -lysine acetylation in eubacteria and that KAc of proteins is tightly regulated and crucial in bacterial metabolism (Guan and Xiong, 2011). The accumulated data indicate that KAc may also play a role in bacterial virulence and survival under stress conditions (Ma and Wood, 2011; Lima et al., 2011), furthermore, bacterial siderophore synthesis that is critical for bacterial virulence in the host has been shown to be regulated via post-translational protein acetylation in M. tuberculosis (Vergnolle et al., 2013). Other proteins involved in persistence and virulence are shown to be lysine acetylated in this study. Further studies on the effect of lysine acetylation of these proteins upon persistence, virulence and underlying regulatory network will shed more lights on the biology of M. tuberculosis.


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Conflict of interest The authors have declared no conflict of interest. Acknowledgements This work was supported by New Century Excellent Talents in Universities [grant number NCET-11-0703], National Natural Science Foundation [grant numbers: 81371851, 81071316, 81271882], National Megaprojects for Key Infectious Diseases [grant number 2008ZX10003-006], Excellent PhD thesis fellowship of Southwest University [grant numbers kb2010017, ky2011003], the Fundamental Research Funds for the Central Universities [nos. XDJK2014D040, XDJK2011D006, XDJK2012D011, XDJK2012D007 and XDJK2013D003], The Chongqing Municipal Committee of Education for Postgraduates Excellence program (nos. YJG123104, 2013JY201). All the mycobacterial strains used in this project were acquired from the “Beijing Bio-Bank of clinical resources on Tuberculosis”. References Allfrey VG, Faulkner R, Mirsky AE. Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc Natl Acad Sci U S A 1964;51:786–94. Choudhary C, et al. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 2009;325(5942):834–40. Colak G, et al. Identification of lysine succinylation substrates and the succinylation regulatory enzyme CobB in Escherichia coli. Mol Cell Proteomics 2013;12(12):3509–20. Cole ST, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998;393(6685):537–44. Coros A, et al. The specialized secretory apparatus ESX-1 is essential for DNA transfer in Mycobacterium smegmatis. Mol Microbiol 2008;69(4):794–808. Cowley S, et al. The Mycobacterium tuberculosis protein serine/threonine kinase PknG is linked to cellular glutamate/glutamine levels and is important for growth in vivo. Mol Microbiol 2004;52(6):1691–702. Dessen A, et al. Crystal structure and function of the isoniazid target of Mycobacterium tuberculosis. Science 1995;267(5204):1638–41. Finkemeier I, et al. Proteins of diverse function and subcellular location are lysine acetylated in Arabidopsis. Plant Physiol 2011;155(4):1779–90. Guan KL, Xiong Y. Regulation of intermediary metabolism by protein acetylation. Trends Biochem Sci 2011;36(2):108–16. Guzman M, et al. Dengue: one of the great emerging health challenges of the 21st century. Expert Rev Vaccines 2004;3(5):511–20. Honer zu Bentrup K, Russell DG. Mycobacterial persistence: adaptation to a changing environment. Trends Microbiol 2001;9(12):597–605. Honer Zu Bentrup K, et al. Characterization of activity and expression of isocitrate lyase in Mycobacterium avium and Mycobacterium tuberculosis. J Bacteriol 1999;181(23):7161–7. Hu LI, Lima BP, Wolfe AJ. Bacterial protein acetylation: the dawning of a new age. Mol Microbiol 2010;77(1):15–21. Jeffers V, Sullivan WJ. Lysine acetylation is widespread on proteins of diverse function and localization in the protozoan parasite Toxoplasma gondii. Eukaryot Cell 2012;11(6):735–42. Kim SC, et al. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol Cell 2006;23(4):607–18. Kim D, et al. The acetylproteome of Gram-positive model bacterium Bacillus subtilis. Proteomics 2013;13(10–11):1726–36. Kouzarides T. Acetylation: a regulatory modification to rival phosphorylation? EMBO J 2000;19(6):1176–9. Lee DW, et al. Proteomic analysis of acetylation in thermophilic Geobacillus kaustophilus. Proteomics 2013;13(15):2278–82.

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Global profiling of lysine acetylation in human histoplasmosis pathogen Histoplasma capsulatum.

Quantitative Profiling of Lysine Acetylation Reveals Dynamic Crosstalk between Receptor Tyrosine Kinases and Lysine Acetylation.

Acetylome analysis reveals diverse functions of lysine acetylation in Mycobacterium tuberculosis.

Proteome-wide profiling of protein lysine acetylation in Aspergillus flavus.

Comprehensive profiling of lysine acetylation suggests the widespread function is regulated by protein acetylation in the silkworm, Bombyx mori.

Proteome-wide analysis of lysine acetylation in the plant pathogen Botrytis cinerea.

Lysine ubiquitination and acetylation of human cardiac 20S proteasomes.

Acetylation site specificities of lysine deacetylase inhibitors in human cells.

Chemoproteomic profiling of lysine acetyltransferases highlights an expanded landscape of catalytic acetylation.

Histone 4 lysine 8 acetylation regulates proliferation and host-pathogen interaction in Plasmodium falciparum.

Pathogen roid rage: cholesterol utilization by Mycobacterium tuberculosis.

Stratification of Latent Mycobacterium tuberculosis Infection by Cellular Immune Profiling.

Acetylation of acetyl-CoA synthetase from Mycobacterium tuberculosis leads to specific inactivation of the adenylation reaction.

Enhancement of lysine acetylation accelerates wound repair.

Targeting bromodomains: epigenetic readers of lysine acetylation.

Site-specific reactivity of nonenzymatic lysine acetylation.

Modulation of Central Carbon Metabolism by Acetylation of Isocitrate Lyase in Mycobacterium tuberculosis.

A method to determine lysine acetylation stoichiometries.

The human antibody response to the surface of Mycobacterium tuberculosis.

CBP.

Human leukocyte antigen, tuberculosis, and Mycobacterium tuberculosis-specific antibody.

A random sequential mechanism of aminoglycoside acetylation by Mycobacterium tuberculosis Eis protein.

Mycobacterium tuberculosis replicates within necrotic human macrophages.

Molecular profiling of Mycobacterium tuberculosis identifies tuberculosinyl nucleoside products of the virulence-associated enzyme Rv3378c.