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The Ser/Thr Protein Kinase Protein-Protein Interaction Map of M. tuberculosis*

2017; Elsevier BV; Volume: 16; Issue: 8 Linguagem: Inglês

10.1074/mcp.m116.065771

ISSN

1535-9484

Autores

Fanlin Wu, Yin Liu, He‐wei Jiang, Yizhao Luan, Hainan Zhang, Xianghuo He, Zhaowei Xu, Jingli Hou, Liyun Ji, Zhi Xie, Daniel M. Czajkowsky, Wei Yan, Jiaoyu Deng, Lijun Bi, Xian‐En Zhang, Sheng‐ce Tao,

Tópico(s)

vaccines and immunoinformatics approaches

Resumo

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the leading cause of death among all infectious diseases. There are 11 eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb, which are thought to play pivotal roles in cell growth, signal transduction and pathogenesis. However, their underlying mechanisms of action remain largely uncharacterized. In this study, using a Mtb proteome microarray, we have globally identified the binding proteins in Mtb for all of the STPKs, and constructed the first STPK protein interaction (KPI) map that includes 492 binding proteins and 1,027 interactions. Bioinformatics analysis showed that the interacting proteins reflect diverse functions, including roles in two-component system, transcription, protein degradation, and cell wall integrity. Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan (PG) biosynthesis, e.g. MurC. The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb, thus facilitating drug development and effective control of Mtb. Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the leading cause of death among all infectious diseases. There are 11 eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb, which are thought to play pivotal roles in cell growth, signal transduction and pathogenesis. However, their underlying mechanisms of action remain largely uncharacterized. In this study, using a Mtb proteome microarray, we have globally identified the binding proteins in Mtb for all of the STPKs, and constructed the first STPK protein interaction (KPI) map that includes 492 binding proteins and 1,027 interactions. Bioinformatics analysis showed that the interacting proteins reflect diverse functions, including roles in two-component system, transcription, protein degradation, and cell wall integrity. Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan (PG) biosynthesis, e.g. MurC. The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb, thus facilitating drug development and effective control of Mtb. Tuberculosis (TB)1 is now the leading cause of death from infectious diseases, with more than 1.5 million deaths just in 2014 (1.WHO Global tuberculosis report 2015.Global tuberculosis report. 2015; Google Scholar). With nearly 10 million newly identified cases in 2014, and the rapid emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb), a causative agent of tuberculosis, the threat of this disease to public health worldwide continues to grow substantially (2.Horsburgh Jr, C.R. Barry 3rd, C.E. Lange C. Treatment of Tuberculosis.N. Engl. J. Med. 2015; 373: 2149-2160Crossref PubMed Scopus (221) Google Scholar). There is thus an urgent need for effective vaccines and drugs to reduce the global burden of TB. One of the most significant reasons for the slow progress in discovering and characterizing appropriate vaccine candidates and drug targets has been the general lack of knowledge about basic biological pathways in Mtb and how they may be regulated. Phosphorylation is widely recognized to play a significant role in signal transduction in many pathways in both eukaryotes and prokaryotes (3.Newman R.H. Hu J. Rho H.S. Xie Z. Woodard C. Neiswinger J. Cooper C. Shirley M. Clark H.M. Hu S. Hwang W. Jeong J.S. Wu G. Lin J. Gao X. Ni Q. Goel R. Xia S. Ji H. Dalby K.N. Birnbaum M.J. Cole P.A. Knapp S. Ryazanov A.G. Zack D.J. Blackshaw S. Pawson T. Gingras A.C. Desiderio S. Pandey A. Turk B.E. Zhang J. Zhu H. Qian J. Construction of human activity-based phosphorylation networks.Mol. Syst. Biol. 2013; 9: 655Crossref PubMed Scopus (135) Google Scholar). Serine/threonine protein kinases (STPKs) are one of the major groups of protein kinases in eukaryotes. In contrast to many of the widely studied bacterial pathogens and model organisms that have few or no STPKs but many two-component systems, the Mtb genome encodes 11 STPKs (4.Dworkin J. Ser/Thr phosphorylation as a regulatory mechanism in bacteria.Curr. Opin. Microbiol. 2015; 24: 47-52Crossref PubMed Scopus (95) Google Scholar). According to their sequence similarity, these STPKs have been grouped into five clades, namely Clade I (pknA, pknB, pknL), Clade II (pknD, pknE, pknH), Clade III (pknF, pknI, pknJ), Clade IV (pknG) and Clade V (pknK) (5.Narayan A. Sachdeva P. Sharma K. Saini A.K. Tyagi A.K. Singh Y. Serine threonine protein kinases of mycobacterial genus: phylogeny to function.Physiol. Gen. 2007; 29: 66-75Crossref PubMed Scopus (74) Google Scholar). It is known that these STPKs play critical roles in adaptation to various environmental conditions (6.Ortega C. Liao R. Anderson L.N. Rustad T. Ollodart A.R. Wright A.T. Sherman D.R. Grundner C. Mycobacterium tuberculosis Ser/Thr protein kinase B mediates an oxygen-dependent replication switch.PLoS Biol. 2014; 12: e1001746Crossref PubMed Scopus (57) Google Scholar), cell wall synthesis (7.Khan S. Nagarajan S.N. Parikh A. Samantaray S. Singh A. Kumar D. Roy R.P. Bhatt A. Nandicoori V.K. Phosphorylation of enoyl-acyl carrier protein reductase InhA impacts mycobacterial growth and survival.J. Biol. Chem. 2010; 285: 37860-37871Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), cell division (8.Jani C. Eoh H. Lee J.J. Hamasha K. Sahana M.B. Han J.-S. Nyayapathy S. Lee J.-Y. Suh J.-W. Lee S.H. Rehse S.J. Crick D.C. Kang C.-M. Regulation of polar peptidoglycan biosynthesis by Wag31 phosphorylation in mycobacteria.BMC Microbiol. 2010; 10: 327Crossref PubMed Scopus (83) Google Scholar) and pathogenicity (9.Wolff K.A. de la Pena A.H. Nguyen H.T. Pham T.H. Amzel L.M. Gabelli S.B. Nguyen L. A redox regulatory system critical for mycobacterial survival in macrophages and biofilm development.PLoS Pathogens. 2015; 11: e1004839Crossref PubMed Scopus (70) Google Scholar). However, except for a few relatively well-studied STPKs, such as PknA, PknB (10.Gee C.L. Papavinasasundaram K.G. Blair S.R. Baer C.E. Falick A.M. King D.S. Griffin J.E. Venghatakrishnan H. Zukauskas A. Wei J.R. Dhiman R.K. Crick D.C. Rubin E.J. Sassetti C.M. Alber T. A phosphorylated pseudokinase complex controls cell wall synthesis in mycobacteria.Sci. Signal. 2012; 5: ra7Crossref PubMed Scopus (127) Google Scholar, 11.Kang C.M. Nyayapathy S. Lee J.Y. Suh J.W. Husson R.N. Wag31, a homologue of the cell division protein DivIVA, regulates growth, morphology and polar cell wall synthesis in mycobacteria.Microbiology-Sgm. 2008; 154: 725-735Crossref PubMed Scopus (191) Google Scholar), and PknG (12.Walburger A. Koul A. Ferrari G. Nguyen L. Prescianotto-Baschong C. Huygen K. Klebl B. Thompson C. Bacher G. Pieters J. Protein kinase G from pathogenic mycobacteria promotes survival within macrophages.Science. 2004; 304: 1800-1804Crossref PubMed Scopus (444) Google Scholar), these STPKs are poorly characterized. Further, how these STPKs function on a systems-wide level is completely unknown. To understand the roles of STPKs systematically, a highly effective first step is the characterization of the global protein-protein interactions (PPIs) in which the STPKs are involved. Although there are a handful of known STPK-protein interactions (KPIs) in Mtb, e.g. PknG and GarA (13.O'Hare H.M. Duran R. Cervenansky C. Bellinzoni M. Wehenkel A.M. Pritsch O. Obal G. Baumgartner J. Vialaret J. Johnsson K. Alzari P.M. Regulation of glutamate metabolism by protein kinases in mycobacteria.Mol. Microbiol. 2008; 70: 1408-1423Crossref PubMed Scopus (132) Google Scholar), which regulates tricarboxylic acid cycle and glutamate synthesis, PknB and Wag31 (14.Hamasha K. Sahana M.B. Jani C. Nyayapathy S. Kang C.M. Rehse S.J. The effect of Wag31 phosphorylation on the cells and the cell envelope fraction of wild-type and conditional mutants of Mycobacterium smegmatis studied by visible-wavelength Raman spectroscopy.Biochem. Biophys. Res. Commun. 2010; 391: 664-668Crossref PubMed Scopus (15) Google Scholar), a component for cell division, PknB and a maltosyltransferase GlgE (15.Leiba J. Syson K. Baronian G. Zanella-Cleon I. Kalscheuer R. Kremer L. Bornemann S. Molle V. Mycobacterium tuberculosis maltosyltransferase GlgE, a genetically validated antituberculosis target, is negatively regulated by Ser/Thr phosphorylation.J. Biol. Chem. 2013; 288: 16546-16556Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). These known KPIs are far too few to enable a systems-wide understanding of the roles of STPKs in Mtb. Protein microarray is a powerful tool for proteome-wide study (16.Zhu H. Bilgin M. Bangham R. Hall D. Casamayor A. Bertone P. Lan N. Jansen R. Bidlingmaier S. Houfek T. Mitchell T. Miller P. Dean R.A. Gerstein M. Snyder M. Global analysis of protein activities using proteome chips.Science. 2001; 293: 2101-2105Crossref PubMed Scopus (1935) Google Scholar), including those that delineate global PPIs (17.Fasolo J. Sboner A. Sun M.G. Yu H. Chen R. Sharon D. Kim P.M. Gerstein M. Snyder M. Diverse protein kinase interactions identified by protein microarrays reveal novel connections between cellular processes.Genes Develop. 2011; 25: 767-778Crossref PubMed Scopus (53) Google Scholar, 18.Chen Y. Yang L.N. Cheng L. Tu S. Guo S.J. Le H.Y. Xiong Q. Mo R. Li C.Y. Jeong J.-S. Jiang L. Blackshaw S. Bi L.J. Zhu H. Tao S.C. Ge F. Bcl2-associated athanogene 3 interactome analysis reveals a new role in modulating proteasome activity.Mol. Cell. Proteomics. 2013; 12: 2804-2819Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). To accelerate our understanding of Mtb and tuberculosis, an Mtb proteome microarray, carrying most of the Mtb proteins, has recently been constructed particularly for the characterization of PPIs (19.Deng J. Bi L. Zhou L. Guo S.J. Fleming J. Jiang H.W. Zhou Y. Gu J. Zhong Q. Wang Z.X. Liu Z. Deng R.P. Gao J. Chen T. Li W. Wang J.F. Wang X. Li H. Ge F. Zhu G. Zhang H.N. Gu J. Wu F.L. Zhang Z. Wang D. Hang H. Li Y. Cheng L. He X. Tao S.C. Zhang X.E. Mycobacterium tuberculosis proteome microarray for global studies of protein function and immunogenicity.Cell Reports. 2014; 9: 2317-2329Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Herein, we applied the Mtb proteome microarray to identify the roles of the Mtb STPKs on a systems-wide level. Overall, we obtained a KPIs network of 492 binding proteins and 1027 interactions, which represents the first and the most comprehensive PPIs network of STPKs in Mtb. Bioinformatic analysis revealed that cell wall related activities were highly enriched in this network, whereas functional analysis confirmed this observation and demonstrated that PknG can regulate Mtb cell wall biogenesis through interaction with the Mur ligase MurC. We believe that the STPK-KPIs network presented here will become an invaluable resource in future biological and clinical studies of Mtb. M. tuberculosis protein microarrays containing about 4200 full-length GST fusion proteins spotted in duplicate were used in this study (19.Deng J. Bi L. Zhou L. Guo S.J. Fleming J. Jiang H.W. Zhou Y. Gu J. Zhong Q. Wang Z.X. Liu Z. Deng R.P. Gao J. Chen T. Li W. Wang J.F. Wang X. Li H. Ge F. Zhu G. Zhang H.N. Gu J. Wu F.L. Zhang Z. Wang D. Hang H. Li Y. Cheng L. He X. Tao S.C. Zhang X.E. Mycobacterium tuberculosis proteome microarray for global studies of protein function and immunogenicity.Cell Reports. 2014; 9: 2317-2329Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Microarrays were blocked for 1 h at RT with shaking in blocking buffer (1× PBS at pH 7.4, 5% BSA, 0.1% Tween-20). Purified V5 tagged kinases probe was diluted in probing buffer (1× PBS, 0.1% Tween-20, 1% BSA). After blocking, the microarrays were incubated in 3 ml of the diluted kinase pool at a final concentration of 10–50 μg/ml at 4 °C overnight. The arrays were then washed 3 times, 10 min each in 25 ml of PBST (1× PBS at pH 7.4, 0.1% Tween-20) buffer at RT with shaking at 60 rpm. The microarrays were then probed with 1 ml of anti-V5 antibody (Sigma-Aldrich, Saint Louis, MI) at a concentration of 260 ng/ml in PBST buffer at RT for 1 h. Following antibody detection, the microarrays were probed with a cy5-conjugated antibody (Jackson Immuno Research, West Grove, PA) at a concentration of 260 ng/ml in PBST buffer at RT for 1 h. The microarrays were washed in 25 ml of PBST buffer for 3 times 10 min each and air dried. The microarray results were recorded using a 4200AL microarray scanner (Molecular Devices, Abingdon, UK). The image file was processed using Genepix software (Molecular Devices). The process of data analysis includes four steps: (1) background correction, (2) normalization, (3) identification of positive hits, and (4) removal of nonspecific interactions. The first step, background correction, is critical to reduce background noise. Next, we quantified the signal intensity of each protein on the array through dividing the median foreground intensity by the median background. To identify positive hits on the microarrays, we estimated the standard deviation from the signal intensity distribution, as described in detail previously (20.Hu S. Xie Z. Onishi A. Yu X. Jiang L. Lin J. Rho H.S. Woodard C. Wang H. Jeong J.S. Long S. He X. Wade H. Blackshaw S. Qian J. Zhu H. Profiling the human protein-DNA interactome reveals ERK2 as a transcriptional repressor of interferon signaling.Cell. 2009; 139: 610-622Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). A cutoff of four standard deviations above the mean was chosen in this study. Since each protein was printed in duplicate, a protein was scored as a positive hit only if both spots of the same protein showed signal intensity higher than a cutoff value determined for that microarray experiment. The binding kinetics of kinase and their interactors were measured using a ForteBio 70 Octet system. Affinity purified kinases PknB, PknG, PknD and PknH was biotinylated using an EZ-Link Sulfo-NHS-LC-Biotin protein biotinylation kit (Thermo Scientific, Bremen, Germany) according to the manufacturer's instructions. Biotinylated kinases were tethered on the tip surface of a streptavidin-coated sensor. The binding partner in S.D. buffer (1 × PBS, pH 7.4 with 0.02% Tween-20 and 0.1% BSA) was then exposed to tethered biotinylated kinases and binding was measured by coincident change in the interference pattern. SSA tips (Pall ForteBio, Menlo Park, NJ) were prewet in S.D. buffer, which served as the background buffer for the immobilization. This involved establishing a stable baseline (60 s), loading a 50 μg/ml kinase (300 s), balancing tips with S.D. buffer (60 s), association with 500 mm substrate (dissolved in S.D. buffer) (300 s) and then eluting nonspecific binding with S.D. buffer (300 s). Final immobilization levels were 1.0 ± 0.2 nm. Data was analyzed using the ForteBio Data Analysis Software 7. The data was fit to a 1:1 binding model to calculate an association and dissociation rate, and KD was calculated using the ratio Kd/Ka. The PknG coding ORF sequence and PknB, PknD, PknH kinase domain sequences were cloned into the bait vector pGBKT7, and the interactors coding sequences into the prey vector pGADT7, which was modified into a Gateway system. 96 gene plasmids of interacting proteins with the BP clones were selected from our Mtb BP clone library, and straightly joined to the prey vector with one step of LR reaction. Y2H interaction assays were performed using the Matchmaker 2-hybrid system (Takara Bio, Tokyo, Japan) according to the manufacturer's instructions. The two recombinant plasmids were cotransformed to the Yeast strains (AH109) and grown on the medium of S.D./-Leu, -Trp for selecting the positive transformants. And then with the S.D./-Leu, -Trp, -His, -Ade medium to select the interactors. The mycobacterial protein fragment complementation (MPFC) assay was performed as described (21.Singh A. Mai D. Kumar A. Steyn A.J. Dissecting virulence pathways of Mycobacterium tuberculosis through protein-protein association.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11346-11351Crossref PubMed Scopus (117) Google Scholar). The genes of interest were PCR-amplified and cloned into pUAB300, PknB and PknD were cloned into pUAB400, whereas pUAB100 (expressing mDHFR fragment F1, 2) and pUAB200 (expressing mDHFR fragment F3) were set as positive controls. M. smegmatis (Msm) was cotransformed with both plasmids; the transformants were selected on 7H10 agar plates with 25 μg/ml kanamycin and 50 μg/ml hygromycin and tested for growth over 4 days on 7H10 kanamycin/hygromycin plates supplemented with 20 μg/ml trimethoprim (TMP). The four kinases (PknB, PknD, PknG and PknH) were cloned to pET28a and expressed in E. coli BL21 (DE3). While the interaction proteins (15 substrates/kinase) were constructed with vector pDEST15 using the Gateway system and expressed in E. coli BL21 (DE3). The STPKs and the interacting proteins were purified with Ni2+ Sepharose beads and GST-Sepharose beads, respectively, according to the manufacturer's instruction. The proteins were quantified with silver staining (Beyotime, Nantong, China). In vitro phosphorylation was performed as described (15.Leiba J. Syson K. Baronian G. Zanella-Cleon I. Kalscheuer R. Kremer L. Bornemann S. Molle V. Mycobacterium tuberculosis maltosyltransferase GlgE, a genetically validated antituberculosis target, is negatively regulated by Ser/Thr phosphorylation.J. Biol. Chem. 2013; 288: 16546-16556Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Briefly, the reaction is composed of 50 μg substrate in 200 μl of kinase buffer (50 mm Tris-HCl, pH 7.0, 1 mm DTT, 5 mm MgCl2, 2 mm MnCl2) with 5 mm ATP, and 10 μg kinase, and a control phosphorylation assay without ATP was included for each of the selected KPIs. As a sum, 60 substrates were carried out for kinase reaction and followed with 60 control reaction. The reactions were carried out for 2 h at 37 °C. The phosphorylation events were then read out by immunoblot with an anti-ser/thr phosphorylation antibody (Abcam, Cambridge, USA). The experiments were carried out twice. After phosphorylation reactions, to obtain high efficiency for MS analysis, >10 samples were grouped as one. In total, 8 groups were prepared. The grouped samples were concentrated to a protein adsorption film (PALL, Port Washington, NY), washed with 50 mm ammonium bicarbonate for 3 times at 4 °C centrifuged at 10,000 × g for 20 min, and reduced with carboxyamidomethyl at 60 °C for 30 min. Proteins were digested with sequencing grade modified trypsin (Promega, Madison, WI) at 37 °C for 16 h - 24 h. The tryptic digested protein sample was eluted from the membrane with 50 mm ammonium bicarbonate for 2 times, and then dried by lyophilization. Phosphopeptides from digested peptides were enriched by using Phosphopeptide Enrichment TiO2 Mag Sepharose (GE healthcare life sciences, Parramatta, Australia) according to the manufacturer's instruction. Briefly, the dried peptides were redissolved in 200 μl TiO2 phospho binding buffer containing 1 m glycolic acid in 80% acetonitrile, 5% trifluoroacetic acid and then mixed with 50 μl TiO2 phospho binding Resin. After 1 h incubation, the supernatant was discarded, and TiO2 resin was washed 3 times with the wash buffer (80% acetonitrile, 1% trifluoroacetic acid). After that, the phosphopeptides were eluted by adding 50 μl elution buffer (5% ammonium hydroxide, pH ∼ 12). The eluted fractions were combined and dried by lyophilization and reconstituted in 20 μl of 0.1% formic acid (FA) for LC-MS/MS analysis (22.Yang M.K. Qiao Z.X. Zhang W.Y. Xiong Q. Zhang J. Li T. Ge F. Zhao J.D. Global phosphoproteomic analysis reveals diverse functions of serine/threonine/tyrosine phosphorylation in the model cyanobacterium Synechococcus sp. strain PCC 7002.J. Proteome Res. 2013; 12: 1909-1923Crossref PubMed Scopus (61) Google Scholar). The phosphorylation peptides were analyzed by online nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS) on a Q Exactive Plus quadrupole orbitrap mass spectrometer (Thermo fisher Scientific). Samples (1 μl) were loaded by an autosampler onto a 2 cm packed pre-column (75 μm ID × 360 μm OD) in 0.1% HCOOH/water (buffer A) at a flow rate of 1 μl/min for 5 min. Analytical separation was performed over a 15 cm packed column (75 μm ID x 360 μm OD) at 300 nl/min with a 60 min gradient of increasing CH3CN (buffer B, 0.1% HCOOH/CH3CN). Both precolumn (5 μm diameter, 200 Å pore size) and analytical column (3 μm diameter, 100 Å pore size) were packed with C18-reversed phase silica (DIKMA-inspireTM) using a pressure bomb. Following sample loading, buffer B was increased rapidly from 3% to 6% over 5 min and then shallowly to 22% over 36 min, and then to 35% over 9 min followed by a quick increase to 95% over 3 min, and hold at 95% for 7 min. The total acquisition duration lasted for 60 min. Survey full scan MS spectra (m/z 350–1800) were acquired in the Orbitrap with 70 000 resolution (m/z 200) after accumulation of ions to a 3 × 106 target value based on predictive AGC from the previous full scan. Dynamic exclusion was set to 60 s. The raw files were processed using the LC/MS software Data Analysis 4.0 (Bruker Compass software). For the improved identification of phosphopeptides, all MS/MS samples were analyzed using MASCOT 2.3 (23.Fu Y. Yang Q. Sun R. Li D. Zeng R. Ling C.X. Gao W. Exploiting the kernel trick to correlate fragment ions for peptide identification via tandem mass spectrometry.Bioinformatics. 2004; 20: 1948-1954Crossref PubMed Scopus (96) Google Scholar). MASCOT were set up to search a database of M.tuberculosis database from SwissProt (http://web.expasy.org/docs/swiss-prot_guideline.html) containing 2067 annotated protein sequences. The search criteria were as follows: trypsin digestion; carbamidomethylation (Cys) was set as fixed modification, whereas oxidation (M), phospho (ST), and phospho (Y) were considered as variable modifications; and two missed cleavages were allowed. Allowed maximum mass deviation was 0.4 Da (monoisotopic) for the precursor ion and fragment maximum mass deviation was 0.6 Da (monoisotopic). For results searching, the peptide-spectrum matches (PSMs) were filtered based on the score threshold of a 1% false discovery rate (FDR), according to the formula: FDR = 2[nDecoy/(nDecoy + nTarget)] (24.Macek B. Mijakovic I. Olsen J.V. Gnad F. Kumar C. Jensen P.R. Mann M. The serine/threonine/tyrosine phosphoproteome of the model bacterium Bacillus subtilis.Mol. Cell. Proteomics. 2007; 6: 697-707Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). And also, all fragmentation spectra of the phosphorylated peptides assigned to the corresponding proteins were manually evaluated and identified according to the neutral loss of ion MH+-80. Finally, to pinpoint the actual phosphorylation site within the identified peptide, the localization probabilities for phosphorylation sites were calculated from the posttranslational modification score algorithm with PhosphoRS analysis using Protein Discovery software (Thermo). Phosphorylation sites that were occupied with a probability > 0.95 were identified as phosphorylation sites. Bacteria harboring pTetInt-PknG, pGrna-MurC pGrna-MurC was grown at 37 °C with shaking at 200 rpm overnight to OD600 = 0.6. And then induced with anhydrote-tracycline (ATc) at concentrations of 20 ng/μl for 24 h to harvested. The supernatant was resuspended with PBS, and lyzed through high pressure cracker (Union-Biotech, Shanghai, China). Lysate was centrifuged to separate the soluble and insoluble fractions. Soluble fractions were used for coimmunoprecipitation. Briefly, mouse anti-flag antibody (Sigma-Aldrich, St. Louis, MO) was added to 2 mg of soluble lysate (1:100 dilution), and the mixture was incubated at 4 °C with shaking for overnight. Affi-Gel protein G agarose resin (Roche, Basel, Switzerland) was then added to the lysate and incubated at at 4 °C with shaking for 4 h to bind to the antibody-antigen complexes. The resin was then washed with PBST for 6 times, and SDS-PAGE loading buffer (Beyotime, Nantong, China) was added. The resulting samples were resolved on SDS-PAGE and transferred onto a nitrocellulose membrane for Western blot analysis with the rabbit anti-PknG antibodies. The MurC reaction mixture (100 μl) contains 50 mm Bis-tris propane buffer (pH 7.0), 10 mm MgCl2, 10 mm KCl, 1 mm UDP-GlcNAc, 1 mm PEP, 1 mm NADPH, 2 mm l-Ala, 2 mm ATP and 200 μg cell lysate. The reaction was performed at 37 °C for 1 h and terminated by the addition of 500 μl methanol for mass spectrometry identification. LC-HRMS was performed as described previously (Vogliardi et al., 2011) on a Waters ACQUITY UPLC system equipped with a binary solvent delivery manager and a sample manager, coupled with a Waters Micromass Q-TOF Premier Mass Spectrometer equipped with an electrospray interface (Waters Corporation, Milford, MA). Briefly, LC was performed on a Syncronis HILIC column (50 × 2.1 mm, 1.7 μm). The column was eluted with 200 mm ammonium formate aqueous solution and acetonitrile in gradient mode at a flow rate of 0.30 ml/min at 30 °C. MS was performed using negative polarity, 2.4KV capillary voltage, 30 V sampling cone, 4eV collision energy, source temperature of 110 °C, and a desolvation temperature of 350 °C. The flow rate for the desolvation gas was set at 600 L/h. Scan range was set to m/z 50∼1000, scan time to 0.3 s and interscan time to 0.02 s. The Msm strains were grown to log phase and 100 μl of culture were spread onto a glass slide. The slides were heated at 100 °C for 2 min, dipped into 10% formalin for 30 min, dried and stained using the TB Fluorescent Stain Kit M (BD, Franklin Lakes, NJ) or the TB Stain Kit (BD) according to the manufacturer's instructions. For biofilm cultures grown on liquid medium, 10 ml of biofilm medium with a modified version of M63 in a 90 × 15 mm PVC Petri dish was inoculated with 10 μl of a saturated culture and incubated at 30 °C without disturbance (25.Ojha A. Anand M. Bhatt A. Kremer L. Jacobs Jr, W.R. Hatfull G.F. GroEL1: a dedicated chaperone involved in mycolic acid biosynthesis during biofilm formation in mycobacteria.Cell. 2005; 123: 861-873Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). The overall design of this study is shown in Fig. 1A. Briefly, the study was composed of 4 parts: preparation of the Mtb proteome microarray and the functional kinases (Fig. 1B), screening KPIs on the microarray and validation (Fig. 1C), bioinformatics analysis and KPIs map construction (Fig. 1D), and functional studies of select, newly discovered KPIs. To systematically identify STPK-KPIs, we used the newly constructed Mtb proteome microarray, which contains 4262 unique Mtb proteins (19.Deng J. Bi L. Zhou L. Guo S.J. Fleming J. Jiang H.W. Zhou Y. Gu J. Zhong Q. Wang Z.X. Liu Z. Deng R.P. Gao J. Chen T. Li W. Wang J.F. Wang X. Li H. Ge F. Zhu G. Zhang H.N. Gu J. Wu F.L. Zhang Z. Wang D. Hang H. Li Y. Cheng L. He X. Tao S.C. Zhang X.E. Mycobacterium tuberculosis proteome microarray for global studies of protein function and immunogenicity.Cell Reports. 2014; 9: 2317-2329Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The proteome microarrays were freshly prepared to ensure their quality. To facilitate expression and purification, all 11 Mtb STPKs were cloned into the expression vector pEGH-A with an additional C-terminal V5 epitope. The kinases were then affinity purified through an N-terminal GST tag. Silver staining clearly demonstrated that all the 11 STPKs were of right size and successfully purified (Fig. 1B. To verify that the expressed kinases are active, we performed an auto-phosphorylation assay with ATP for each kinase (15.Leiba J. Syson K. Baronian G. Zanella-Cleon I. Kalscheuer R. Kremer L. Bornemann S. Molle V. Mycobacterium tuberculosis maltosyltransferase GlgE, a genetically validated antituberculosis target, is negatively regulated by Ser/Thr phosphorylation.J. Biol. Chem. 2013; 288: 16546-16556Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), and a control without ATP was also included. The reactions were then readout using an anti-serine/threonine phosphorylation antibody (Fig. 1B. Significantly higher signals were observed for most of the kinases from the reactions with ATP, compared with that of the reactions without ATP, indicated that the purified kinases are of good activity. For kinases with lower activity, i.e. PknF, PknJ, and PknL, relatively higher concentration of these kinases were included for probing on the Mtb proteome microarray. For each kinase, we probed 2 microarrays, along with a negative control following the same probing procedure except without the kinase. To identify the positive KPIs, the microarray results were extracted by Gene-Pix 6.1 and subjected to scoring by an algorithm designed to measure the relative signal intensity of each protein spop. (20.Hu S. Xie Z. Onishi A. Yu X. Jiang L. Lin J. Rho H.S. Woodard C. Wang H. Jeong J.S. Long S. He X. Wade H. Blackshaw S. Qian J. Zhu H. Profiling the human protein-DNA interactome reveals ERK2 as a transcriptional repressor of interferon signaling.Cell. 2009; 139: 610-622Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). After normalization within each chip to eliminate spatial artifacts, a cutoff value of four standard deviations above the mean intensity was applied, as previously described (20.Hu S. Xie Z. Onishi A. Yu X. Jiang L. Lin J. Rho H.S. Woodard C. Wang H. Jeong J.S. Long S. He X. Wade H. Blackshaw S. Qian J. Zhu H. Profiling the human protein-DNA interac

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