A previously described hyper-virulent clinical Beijing genotype Mycobacterium tuberculosis isolate, SAW5527, isolated from a TB patient attending a primary health care clinic in the Western Cape province, South Africa was used for this phosphoproteomics analysis (de Souza et al., 2010). Secondary cultures were inoculated into 50 ml 7H9 Middlebrooks medium supplemented with Dextrose and Catalase and incubated at 37°C until early-logarithmic phase (OD₆₀₀ between 0.6 and 0.7). Mycobacterial cells were collected by centrifugation (2000 × g for 10 min at 4°C) and washed two times with cold lysis buffer containing 10 mM Tris-HCl (pH 7.4), 0.1% Tween-80, Complete Protease inhibitor cocktail (Roche, Mannheim Germany) and Phosphatase inhibitor cocktail (Roche, Mannheim Germany). An equal amount of 0.1 mm glass beads (Biospec Products Inc., Bartlesville, OK) was added to the cell pellet after centrifugation together with cold 300 μl lysis buffer and 10 μl DNaseI (2U/ml) (NEB, New England Laboratories). Lysis was achieved by mechanical bead-beating in a Rybolyser (Bio101 SAVANT, Vista, CA) for 6 cycles of 20 s at a speed of 4.0 m.s⁻¹, with 1 min cooling periods on ice. The whole cell lysates were filter-sterilized with a sterile 0.22 μm pore acrodisc 25 mm PF syringe filter (Pall Life Sciences, Pall Corporation, Ann Arbour, MI) and stored at −80°C. The protein concentration of the whole cell lysate was determined using the RC DC Protein assay according to manufacturer's instructions (BioRad). A single biological replicate was analyzed in triplicate for downstream phosphoproteomic analysis.

Four milligrams of concentrated whole cell lysate proteins was heated in 4% SDS buffer and 0.1 M dithiothreitol (DTT) in 100 mM Tris/HCl pH 7.5. The samples were processed using Filter Aided Sample Preparation (FASP) (Wiśniewski et al., 2009). In brief, 4 mg dried whole cell lysate protein was resuspended in 250 μl of urea (UA) and loaded onto a 15 ml Amicon filtration device (30 kDa MWCO) and centrifuged at 2000 × g for 40 min at 25°C. After centrifugation, the flow-through was collected in a clean falcon tube and discarded. The concentrated whole cell lysate proteins in the filter unit were diluted in 2 ml 8 M Urea in 0.1 M Tris/HCl (pH 8.5) and re-centrifuged to remove the SDS. The flow-through was discarded and the remaining proteins in the filter unit were alkylated by mixing with 1.5 ml 50 mM iodacetamide (IAA) and incubated in the dark for 20 min to irreversibly modify cysteine. The alkylated proteins were equilibrated with 2 ml 50 mM ammonium bicarbonate (ABC) and digested with trypsin (Promega) in a protein to enzyme ratio of 100:1 at 37°C overnight. After trypsin digestion the filter unit is transferred to a clean collection tube and the peptides were eluted by centrifuged at 14 000 × g for 10 min. The eluted peptides were diluted in 50 μl water to avoid desalting for further processing of the peptide and acidified with trifluoroacetic acid (TFA).

Extracted trypsin digested peptides were diluted to a volume of 7 ml in Solvent A (5 mM monopotassium phosphate (KH₂PO₄) 30% acetonitrile (ACN), pH 2.7). The pH of the diluted peptide samples was adjusted to 2.7 and made up to a volume of 10 ml with 100% ACN. The respective peptide samples were then separated at pH 2.7 by strong cation exchange (SCX ) by loading each peptide mixture onto a cation exchanger column (3.0 mm × 20 cm) (Poly LC, Columbia, MD) containing 5 μm polysulfoethyl aspartamine beads with a 200 Å pore size and a flow rate of 350 μl/min⁻¹ equilibrated with SCX solvent A. The flow-through was collected and the bound peptides were eluted from the columns using an increasing salt gradient (0–30%) containing 5 mM KH₂PO₄ pH and 150 mM KCl. A total of 9 fractions were collected; 5 fractions generated by SCX based on UV absorbance (220 and 280), 3 from the flow-through and 1 salvage fraction (containing washes from the cation exchanger column) from the SCX column as an additional fraction.

All nine fractions (5 SCX, 3 SCX flow-through, 1 salvage fraction) were subjected to Titanium dioxide (TiO₂) phosphopeptide enrichment. The TiO₂-beads,10 μm in size, (GL Sciences, Inc., Japan) was resuspended 30 mg/ml dihydrobenzoic acid (DHB) (Sigma) to prevent non-specific binding. Each of the 9 fractions was incubated 4 times with TiO₂ at a peptide to bead ratio of 1:2–1:8. Each fraction was rotated for 30 min, and then briefly centrifuged (14,000 g × 30 s). The supernatants were aspirated and transferred to a new labeled tube and the phosphopeptides bound to the TiO₂beads were washed twice with 30% ACN and 3% TFA followed by washing twice with 75% ACN and 0.3% TFA. The enriched phosphorylated peptides were eluted with elution buffer containing 25% ammonia and ACN pH10. The eluted phosphopeptides were desalted using in house prepared C₁₈ Stage tips.

The peptides were separated on a column packed in-house with C18 beads (reprosil-AQ Pur, Rd Maisch) on an Proxeon Easy-nLC system (Proxeon Biosystems, Odense, Denmark) using a binary gradient with buffer A (0.5% acetic acid in water) and buffer B (0.5% acetic acid and 80% ACN). The 4 μl of the enriched phosphopeptides from each of the 9 fractions were injected 3 times and were loaded directly without any trapping column with buffer A at a flow rate of 500 nl/min. Elution was carried out at a flow rate of 250 nl/min, with a linear gradient from 10 to 35% buffer B over a period of 95 min followed by 50% buffer B for 15 min. At the end of the gradient the column was washed with 90% buffer B and equilibrated with 5% buffer B for 10 min. The LC system was directly coupled in-line with the LTQ-Orbitrap Velos instrument (Thermo Fisher Scientific) via the Proxeon Biosystems nanoelectrospray source. The mass spectrometer was programmed to acquire in a data-dependant mode with a resolution of 30,000 at 400 m/z with lock mass option enabled for the 445.120025. However, the target lock mass abundance was set to 0% in order to save the injection time for lock mass. For full scans 1E6 ions were accumulated within a maximum injection time of 250 ms in the C trap and detected in the Orbitrap mass analyser. The 10 most intense ions with charge states ≥2 were sequentially isolated and fragmented by high-energy collision dissociation (HCD) mode in the collision cell with normalized collision energy of 40% and detected in the Orbitrap analyser at 75,000 resolution. For HCD based method, the activation time option in the Xcalibur file was set to 0.1 ms. For the high-low strategy, full scans were acquired in the Orbitrap analyser at 60,000 resolution as parallel acquisition is enabled in the high-low mode. Up to the 10 most intense peaks with charge states ≥2 were selected for sequencing to a target value of 5000 with a maximum injection time of 25 ms and fragmented in the ion trap by HCD with normalized collision energy of 35%, activation of 0.25 and activation time of 10 ms.

The raw data acquired were processed using MaxQuant software version (1.4.1.2) and processed as per default workflow. Since HCD spectra were acquired in profile mode, deisotoping was performed similar to survey MS scans to obtain singly charged peak lists and searched against the M. tuberculosis H₃₇Rv protein database (version R11 tuberculist.epfl.ch). The search included cysteine carbamidomethylation as a fixed modification while N-acetylation, oxidation of methionine and phosphorylation at serine, threonine and tyrosine were set as variable modifications. Up to two missed cleavages were allowed for protease digestion and a peptide had to be fully tryptic. Identifications were filtered at 1% FDR at three levels namely; site, peptide and protein using reversed sequences. As such there is no fixed cut-off score threshold but instead spectra were accepted until the 1% false discovery rate (FDR) is reached. Only peptides with a minimum length of 7 amino acids were considered for identification and detected in at least one or more of the replicates. All phosphopeptide spectra were manually validated by applying stringent acceptance criteria: only phosphorylation events on S/T/Y with a localization probability of ≥0.75 and PEP ≤ 0.01 were used for further analysis and reported as high confidence localized phosphosites.

The categorization of identified phosphorylated proteins in terms of functional categorization, molecular function, biological processes and cellular components was carried out using TubercuList-Mycobacterium tuberculosis Database.

In recent years, bacterial S/T/Y kinases and phosphatases have been extensively investigated, with indications that they might play a crucial role in pathogenicity. These Hank's type kinases have the ability to short-circuit the host defense mechanism since they are mostly involved in key biological processes. We identified phosphopeptides from 9 of the 11 STPKs encoded by the M. tuberculosis genome. This included both previously described S/T phosphorylation sites (Boitel et al., 2003; Young et al., 2003; Durán et al., 2005; Villarino et al., 2005; Molle et al., 2006; O'Hare et al., 2008; Prisic et al., 2010; Sajid et al., 2011; Chou et al., 2012; Kusebauch et al., 2014) and novel sites on these STPKs thereby highlighting the complexity of the signal transduction mechanism of this pathogen. Phosphorylated peptides were not detected for PknI and PknK. However MS/MS spectra for these peptides for these proteins were detected with mass spectrometry thereby confirming the presence of these proteins (data not shown).

Of the phosphorylated STPKs, PknA, PknB, and PknG have been shown to be essential for in vitro growth (Sassetti et al., 2003) and to regulate cell growth and cell division and interfere with host signaling pathways (Fernandez et al., 2006). PknE, PknH, PknJ, and PknL have been implicated in the adaptation to the extracellular environment or intracellular survival of M. tuberculosis (Sharma et al., 2006; Lakshminarayan, 2009; Arora et al., 2010; Parandhaman et al., 2014) which is in agreement with reports that during early growth the bacilli undergo a period of adaptation to their external environment (Stock et al., 1989; Soares et al., 2013). PknE is involved in the suppression of apoptosis during nitrate stress (Kumar and Narayanan, 2012) and intracellular survival and adaptation to hostile environments (Parandhaman et al., 2014). In M. tuberculosis, PknH controls the expression of a variety of cell wall related enzymes and regulates in vivo growth in mice (Zheng et al., 2007). PknJ undergoes autophosphorylation and phosphorylates the Thr¹⁶⁸, Thr¹⁷¹, and Thr¹⁷³ residues of Embr (a transcriptional regulator), MmaA4/Hma (a methyltransferase involved in mycolic acid biosynthesis) and PepE (a peptidase located adjacent to the pknJ gene in the M. tuberculosis genome), respectively (Jang et al., 2010). Lastly, PknL is involved in an adaptive response to nutrient starvation. This kinase regulates transcription which allows the bacilli to maintain metabolic activity without sourcing energy from elsewhere (Lakshminarayan, 2009). Furthermore, we identified a number of STPK substrates that were phosphorylated in clinical hyper-virulent M. tuberculosis strain (list not shown) thereby highlighting the complexity of the phosphorylation regulatory network in M. tuberculosis. Even though the role of STPKs in bacterial physiology is not yet fully understood the data presented here could underpin a targeted approach to improving our understanding of STPK-mediated signal transduction mechanisms in M. tuberculosis.

The M. tuberculosis genome encodes for two putative tyrosine phosphatases (PtpA and PtpB) but is not predicted to encode tyrosine kinases (Cole et al., 1998; Bach et al., 2009). Most bacterial phosphorylation sites are on serine and threonine; a survey of 11 bacterial phosphoproteomes revealed that S/T phosphorylation accounted for an average of 48 and 40% of phosphorylated sites, respectively, while tyrosine phosphorylation events account for less than 10% of the overall phosphoproteome (Ge and Shan, 2011). Tyrosine phosphorylated proteins have been previously shown to play important regulatory roles through their involvement in biological functions such as exopolysaccharide production, DNA metabolism, stress responses (Ge et al., 2011; Whitmore and Lamont, 2012). Recently, Kusebauch et al. identified tyrosine phosphorylated proteins in M. tuberculosis and demonstrated that a number of STPKs can phosphorylate tyrosine in either cis or trans (Kusebauch et al., 2014). This suggests that STPKs have the ability to phosphorylate S/T/Y. In this study we identified 13 tyrosine phosphorylation sites in 8 proteins (Table 3). An overlap of three proteins (FhaA, MmpS3, and GroES) and 6 tyrosine phosphorylated sites we similar between this and the previous study (Kusebauch et al., 2014). Five of the tyrosine phosphorylated proteins (FhaA, GroEL2, MmpS3, GroES, and Ppa) identified in this study are essential for in vitro growth (Sassetti et al., 2003; Griffin et al., 2011) and involved in a variety of functions.

This study confirmed and expanded work by Kusebauch et al., where multiple tyrosine phosphopeptides were identified for FhaA. FhaA is a regulatory protein which has been implicated in cell wall biosynthesis (Fernandez et al., 2006) and has a strong association with PknA and PknB (Pallen et al., 2002; Roumestand et al., 2011). We found that the highly S/T/Y phosphorylated FHA-domain contained 6 tyrosine phosphopeptides of which 4 were previously been identified in H₃₇Rv (Kusebauch et al., 2014). FhaA is a major substrate of PknB and has been implicated in the formation of a regulatory complex with MviN required for peptidoglycan biosynthesis (Warner and Mizrahi, 2012). In our dataset, we found that all three of the proteins (PknB, FhaA, and MviN) in the regulator complex were phosphorylated.

We also confirm the presence of a previously reported Y phosphopeptide of MmpS3 and identified a second phosphopeptide. MmpS3 forms part of the mycobacterial membrane protein small family and is an essential protein for mycobacterial growth and cholesterol metabolism (Griffin et al., 2012). The role of phosphorylation of this protein has yet to be determined.

The two proteins GroEL2 and GroES have been identified as potential candidates for antituberculosis treatment (Al-Attiyah et al., 2006). We confirmed the presence of the Y phosphopeptide in GroEL2 identified in H₃₇Rv (Kusebauch et al., 2014). Recently it has been shown that the antigen GroES is sufficient to protect BALB/c mice against challenge infection (Lima et al., 2003) and up-regulated in kanamycin and amikacin resistant isolates (Kumar et al., 2013). Ppa is an inorganic pyrophosphate and is involved in macromolecule biosynthesis. The M. tuberculosis Ppa is highly similar to a well conserved homolog of Legionella. pneumophila PPase which is induced in macrophages, although the M. tuberculosis PPa promotor is not responsive to any specific intracellular triggers (Triccas and Gicquel, 2001).

The identification of virulence factors is crucial in order to improve our understanding of the mechanisms involved in pathogenesis of M. tuberculosis. Several of the phosphorylated virulence factors identified in this study were found to be involved in basic metabolic pathways such a lipid and fatty acid metabolism, secretion systems and response and adaptation to environmental changes. The virulence factor KasB and key enzymes (FadD32, AccD4, and MmaA3) in the mycolic acids biosynthesis pathway were phosphorylated in this hyper-virulent M. tuberculosis strain. The kasB gene is not essential for growth, however, the deletion mutant, ΔkasB, resulted in an alteration in growth morphology and loss of acid-fast staining (Bhatt et al., 2007). This suggests that modification of this protein could influence the synthesis of mycolic acids and thereby the pathogenicity of the bacilli.

The specialized ESX-1 Type VII secretion system (T7SS), unique to pathogenic mycobacteria is responsible for the secretion of two culture filtrate proteins EsxA and EsxB (ESAT-6 and CFP-10). These secretion systems have been shown to be involved in virulence and are critical for intracellular survival (Bitter and Kuijl, 2014) due to their ability to secrete proteins that lack classical signal peptides across the complex cell envelope to host cells during infection (Houben et al., 2014, p. 5). M. tuberculosis have several different ESX regions (ESX-1 to ESX-5) (Daleke et al., 2012) with varying gene numbers and size for each of these secretion machinery. In this study we found 6 T7SS proteins to be phosphorylated in the hyper-virulent strain. In a previous study, proteomics of whole cell extracts of this hyper-virulent M. tuberculosis strain revealed an under-representation of virulence factors such as ESAT-6 and Esx-like proteins (de Souza et al., 2010). The authors showed the abundance of ESAT-6 gene expression was reduced in the hyper-virulent M. tuberculosis suggesting that the low levels of this protein might be as a result of its ability to export these proteins more efficiently into the extracellular environment (de Souza et al., 2010).

The impact of phosphorylation on the functionality of ribosomal proteins is not fully understood. Mikulik et al. hypothesized that phosphorylation of ribosomal proteins induces or stabilizes conformational changes during proteins synthesis which could allow modification of subunit association or changes in interactions with proteins and RNAs (Mikulík et al., 2011). According to the protein phosphorylation database, phosphopeptides of RpmC have been identified in four different bacteria (Soufi et al., 2008; Burnside et al., 2011; Manteca et al., 2011; Misra et al., 2011). The implication of phosphorylation on RpmC has not been investigated. However, in E.coli, RpmC, RplW and Trigger factor are located at the exit tunnel in the ribosome, suggesting that phosphorylation may impact on multiple stages of transcription (Kramer et al., 2002). In our study we identified phosphopeptides for 7 ribosomal proteins. We also identified unique phosphopeptides on ribosomal proteins RpsS and RplX (Table 3).

Twenty-five of phosphorylated proteins identified in our study were also identified in phosphoproteomics studies of other bacteria such as Klebsiella pneumonia (Lin et al., 2009), Helicobacter pylori (Ge et al., 2011), Steptococcus pneumonia (Sun et al., 2010), Bacillus subtillis (Macek et al., 2007), Halobacterium salinarum (Aivaliotis et al., 2009), etc. (Table 4). In our dataset the distribution of S/T/Y seem to be bias toward pT and is in accordance with previously described phosphoproteomes of M. tuberculosis (Prisic et al., 2010; Kusebauch et al., 2014). Manual evaluation of the genome found an over-representation of Threonine relative to Serine (52:48%). This compared to other bacteria such as Acinetobacter baumannii (Soares et al., 2014), Bacillus subtilis (Macek et al., 2007), Escherichia coli (Macek et al., 2008; Soares et al., 2013) and Halobacterium salinarum (Aivaliotis et al., 2009), Pseudomonas aeruginosa (Ravichandran et al., 2009), and Streptomyces coelicolor (Parker et al., 2010) which demonstrate a bias toward pS.

Forty-five of the phosphorylated proteins identified in our study were previously described for M. tuberculosis H₃₇Rv (Boitel et al., 2003; Young et al., 2003; Molle et al., 2004, 2006; Durán et al., 2005; Kang et al., 2005; Villarino et al., 2005; O'Hare et al., 2008; Thakur et al., 2008; Prisic et al., 2010; Sajid et al., 2011; Gee et al., 2012). The reason for not identifying all of the previously identified phosphorylated proteins in the protein phosphorylation database could be ascribed to different genetic backgrounds of the analyzed M. tuberculosis strains, culture conditions, sample preparation and different MS-based proteomics approaches used in each of the studies. Our analysis was performed on a hyper-virulent clinical isolate of M. tuberculosis and a member of the Beijing genotype which is genetically distinct from the laboratory strain M. tuberculosis H₃₇Rv analyzed by Prisic et al. and Kusebauch et al. In addition, the Prisic et al. study reported on the combined phosphoproteome from 6 different conditions (5 different culture conditions and 2 different growth phases) (Prisic et al., 2010) while Kusebauch et al. reported on the phosphoproteome of late-logarithmic phase cultures (Kusebauch et al., 2014), whereas our study analyzed early-logarithmic phase cultures. Even though the overlap between our study of clinical M. tuberculosis and that of the previously described laboratory M. tuberculosis H₃₇Rv is low this work substantially extends our knowledge of the M. tuberculosis phosphoproteome. During logarithmic growth phase of bacterial growth the cells are adapting to the environment of the growth media and biological process such as RNA synthesis, DNA replication and synthesis of micro- and macromolecules are up-regulated. It is important to note that in this study the whole cell lysate proteins were enriched for phosphopeptides and we detected a number of phosphorylated proteins involved in these biological processes such as fatty acid- and lipid biosynthetic metabolism; RNA modification and translation; DNA repair, replication and modification. It is believed that environmental conditions, cell density and growth phase influence the expression of virulence factors by a pathogen (McIver et al., 1995). This is consistent other bacterial phosphoproteomes, thereby emphasizing that S/T/Y phosphorylation is an important process required for the regulation of numerous cellular processes.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.