Lysine succinylation and acetylation in Pseudomonas aeruginosa

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Lysine succinylation and acetylation in Pseudomonas aeruginosa Charlotte Gaviard, Isabelle Broutin, Pascal Cosette, Emmanuelle De, Thierry Jouenne, and Julie Hardouin J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00210 • Publication Date (Web): 17 May 2018 Downloaded from http://pubs.acs.org on May 17, 2018

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Journal of Proteome Research

Lysine succinylation and acetylation in Pseudomonas aeruginosa Charlotte Gaviard1,2, Isabelle Broutin3, Pascal Cosette1,2, Emmanuelle Dé1,2, Thierry Jouenne1,2, and Julie Hardouin1,2 ,* 1

Normandie Univ, UNIROUEN, INSA Rouen, CNRS, PBS, 76000 Rouen, France PISSARO proteomic facility, IRIB, 76821 Mont-Saint-Aignan, France 3 LCRB, UMR 8015 CNRS, University Paris Descartes, Sorbonne Paris City, 75270 Paris cedex 06, France 2

*Correspondence to: Dr Julie Hardouin, Laboratoire Polymères, Biopolymères, Surfaces, UMR CNRS 6270, Université de Rouen, Bâtiment CURIB, 76821 Mont-Saint-Aignan cedex, France. Tel: +33 (0)2 35 14 67 09 E-mail: [email protected]

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ABSTRACT Pseudomonas aeruginosa is a Multi-Drug Resistant human opportunistic pathogen largely involved in nosocomial infections. Unfortunately, effective antibacterial agents lacked. Explore its physiology at the post-translational modifications (PTMs) level may contribute to the renewal of combat tactics. Recently, lysine succinylation was discovered in bacteria and seems to be an interesting PTM. Here, we present the first succinylome and acetylome of P. aeruginosa PA14 cultured in the presence of 4 different carbon sources, using a twodimensional immunoaffinity approach coupled with nano-liquid chromatography tandem mass spectrometry. A total of 1 520 succinylated (612 proteins) and 1 102 acetylated (522 proteins) lysine residues were characterized. Citrate was the carbon source in which we identified the higher number of modified proteins. Interestingly, 622 lysine residues (312 proteins) were observed either acetylated or succinylated. Some of these proteins, were involved in virulence, adaptation, resistance, etc. A label free quantification points out the existence of different protein forms for a same protein (unmodified, succinylated or acetylated) and suggests different abundance in function of the carbon sources. This work is a promising starting point for further investigations on the biological role of lysine succinylation in P. aeruginosa.

Keywords: lysine succinylation, lysine acetylation, Pseudomonas aeruginosa PA14, proteomics


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1. Introduction

Pseudomonas aeruginosa is an ubiquitous Gram-negative bacterium found in all the environments (plants, animals, soil, freshwater, marine, etc). This universal distribution suggests a remarkable degree of genetic and physiological plasticity1. P. aeruginosa is a major human opportunistic pathogen largely involved in nosocomial infections and responsible of a variety of diseases in weakened individuals2. This bacterium is very difficult to eradicate because it is highly resistant to a wide range of antimicrobial agents and able to evade the host defense. It has recently been classified as a critical bacterium and priority number one by the World Health Organization3. Today, it is well known that proteins can perform diverse functions, on short timescales, due to post-translational modifications (PTMs). In contrast to eukaryotes, PTM discovery in bacteria is quite recent. Different kinds of modifications have been described in bacteria4 like the addition of chemical groups on the side chain of specific amino acids (e.g., acetyl, succinyl, methyl) that mainly occurs after protein translation. Proteomics analyses have revolutionized PTMs characterization5 thanks to mass spectrometry improvements, bioinformatics tools and antibody development. Indeed, this strategy has allowed the identification of numerous modified proteins in a single sample. In P. aeruginosa, different studies have been achieved on different PTMs: N-terminal methionine excision6, N-terminal acetylation6, lysine acetylation7 and phosphorylation8–10. Interestingly, some virulence factors were

described

to

be

phosphorylated

and/or

acetylated

(enzymes

involved

in

lipopolysaccharides biosynthesis, elastases LasA and LasB, CbpD, Pvds-regulated endoprotease, aminopeptidase, azurin).It is increasingly shown that many of them play a role in bacterial physiology or virulence11–14. Consequently, the characterization of PTMs is essential to a better understanding of the bacterial virulence, adaptation or/and resistance. Among all amino acid residues, lysine is a fascinating amino acid since its side chain can be modified by a variety of chemical groups. Acetylation is the most investigated lysine PTMs in bacteria7,15–29. Besides, new lysine modifications are currently investigated in bacteria: glutarylation30,

malonylation31,32,

propionylation33–35,

succinylation25,26,36–42.

Lysine

succinylation is increasingly studied and succinylome studies emphasized the frequency and the importance of this PTM within of the prokaryotic kingdom. Interestingly, it was shown that the same lysine of a protein can be acetylated or succinylated25,26,38,42. The addition of these PTMs changes the side chain charge and can thus affect protein conformation, protein interactions, protein sub-cellular localization, and therefore protein function43. In bacteria, little information is available on enzymes involved in addition/removal of PTMs. 3 ACS Paragon Plus Environment


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In the case of lysine acetylation, lysine acetyltransferases and lysine deacetylases add or remove the acetyl group, respectively44. In the case of succinylation, three enzymes are annotated as desuccinylase (AruE) and succinyltransferase (AruF and AruG). They target amino acid ornithine, arginine or glutamate. Unfortunately, no enzyme involved in lysine succinylation or desuccinylation has been described to our knowledge. However, CobB, described first like a NAD+-dependent sirtuin deacetylase, has shown a desuccinylase activity in E. coli36. These two PTMs can also occur non-enzymatically. Indeed, it was previously described that lysine acetylation can be added spontaneously via an acetyl donor, e.g., acetyl CoA45 or acetyl phosphate46–48. Lysine succinylation can be enzyme independent. An increase in succinyl-CoA levels in the bacterial cells can increase this PTM in proteins42,49. Modified proteins and enzymes involved in these mechanisms can be interesting potential targets for the development of new effective treatments50,51 to fight against P. aeruginosa. P. aeruginosa isolates display a high variability in virulence, ranging from very moderate to highly virulent strains. PAO1, which was isolated in 1954 from a wound in Melbourne (Australia)52, is a moderately virulent strain and belongs to a relatively rare clonal group. In contrast, the clinical strain PA14 is a highly virulent isolate and represents the most common clonal group worldwide53. In this study, we will focus on the first repertory of succinylated proteins in P. aeruginosa strain PA14. We also investigated too the lysine acetylome. It was shown that the carbon source influences PTM rate in bacteria25,26,36,41. So therefore, to obtain the most overview of modified proteins in PA14, we did performed the investigation in 4 carbon sources. To this aim, we used a two-dimensional immunoaffinity approach coupled with nanoliquid chromatography mass spectrometry (nanoLC-MS/MS). Overall, a total of 1 520 succinylated sites (612 proteins) and 1 102 acetylated sites (522 proteins) were identified. Citrate was the carbon source conducting attributed to the higher number of modified proteins. Interestingly, we noticed that 622 lysines residues (312 proteins) can be either succinylated or acetylated. Some of these modified proteins are involved in interesting mechanisms, e.g., chemotaxis, efflux pump or bacterial secretion system. This provides the first global succinylation profiling of P. aeruginosa PA14 which may be the starting point for further investigations on the biological role of lysine succinylation in this bacterial species.


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Materials and methods 2.1 Bacterial growth The strain used is P. aeruginosa PA14 obtained from the laboratory of Dr Ausubel (Massachusetts General Hospital). An overnight culture in Mueller Hinton Broth (MHB, Difco) was performed to inoculate 500 mL of Minimal Medium (MM) (Tris-HCl90 mM , Tris-base 4 mM, NH4Cl 9 mM, yeast extract 0,2 %, CaCl2 0,45 mM, MgSO4 0,2 mM, FeSO4 18 µM, MnSO4 18 µM, and glucose 80 mM, sodium citrate 80 mM, sodium succinate 80 mM or sodium glutamate 80 mM) at a final concentration of 107 CFU/mL. Cells were grown at 37 °C under constant shaking (140 rpm) for 24 h. Bacteria were harvested at stationary phases of growth (OD545 = 1.9) by centrifugation (9 000g, 25 min, 4 °C). Bacterial cells were washed three times with 10 mL of Tris-HCl buffer (20 mM, pH 7.4).

2.2 Protein extraction The bacterial pellet was resuspended in 10 ml of 20 mM Tris-HCl buffer supplemented with a protease inhibitor cocktail (20 µL/mL, Protease Inhibitor Cocktail-Bacterial, Sigma-Aldrich) and Histone deacetylase (HDAC) inhibitors (20 mM Nicotinamide (inhibitor of HDAC class III) and 0.3 µM Trichostatin A (inhibitor of HDAC classes I and II) (Sigma Aldrich)). The mixture was freeze-thawed for three cycles and then sonicated on ice six times, each for 1 min. The lysate was centrifuged at 9 000g for 20 min at 4 °C. A part of resulting supernatants was stored in aliquots at -20 °C until further use. An ultracentrifugation was applied to the resulting supernatants (60 000g, 45 min, 4 °C) to separate cytoplasmic and membrane proteins. Protein concentration was evaluated by Bradford analysis (Bio-Rad).

2.3 Sample preparation for enrichment of succinyl-/acetyl-lysine peptides Proteins (3 mg) were solubilized in 6 M urea and 15 mM DTT during 1 h. The reduced proteins were alkylated with 15 mM iodoacetamide during 45 min in the dark, at room temperature. Proteins were precipitated in 4 volumes of ice-cold acetone (-20 °C, 2 h). The protein pellet was resuspended in 1 mL of 10 mM, ammonium bicarbonate (pH 8) and digested with trypsin (enzyme/protein ratio: 1/50) overnight at 37 °C with shaking. Peptides were dried and stored at -20 °C. Peptides enrichments were performed following the manufacturer's instruction (PTM Biolabs). Sixty µL of anti-succinyllysine polyclonal antibodies conjugated to protein A were washed three times with the ETN buffer (1 mM EDTA, 50 mM Tris-base, 100 mM NaCl). Tryptic digests were suspended in ETN buffer, mixed with the conjugated beads, and gently 5 ACS Paragon Plus Environment


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shacked at 4 °C overnight. Beads were washed three times with ETN buffer and three times with water. Peptides were eluted from the beads by three washes with formic acid (FA) 1%. After succinyllysine peptide enrichment, the supernatants were further enriched for acetyllysine peptides using anti-acetyllysine polyclonal antibodies (as described here). After enrichment, peptides were then desalted on C18 tip column, dried and stored at −20 °C until prior to MS analysis. Three biological replicates were run per carbon source

2.4 NanoLC-MS/MS analyses All experiments were performed on a LTQ-Orbitrap Elite coupled to an Easy nLC II system (both from Thermo Scientific). Samples were injected onto an enrichment column (C18 PepMap100, Thermo Scientific). The separation was achieved with an analytical column needle (NTCC-360/100-5-153, NikkyoTechnos). The mobile phase consisted of H2O/FA 0.1% (buffer A) and ACN/FA 0.1% (buffer B). Tryptic peptides were eluted at a flow rate of 300 nL/min, using a three-step linear gradient: from 2 to 40% B over 75 min, from 40 to 80% B in 4 min and at 80% B for 11 min. The mass spectrometer was operated in positive ionization mode with capillary voltage and source temperature set at 1.5 kV and 275 °C, respectively. The samples were analyzed using CID (collision induced dissociation) method. The first scan (MS spectra) was recorded in the Orbitrap analyzer (R = 60,000) with the mass range m/z 400-1800. Then, the 20 most intense ions were selected for MS2 experiments. Singly charged species were excluded for MS2 experiments. Dynamic exclusion of already fragmented precursor ions was applied for 30 s, with a repeat count of 2, a repeat duration of 30 s and an exclusion mass width of ±5 ppm. The precursor isolation width was 2 m/z. Fragmentation occurred in the linear ion trap analyzer with normalized collision energy of 35. All measurements in the Orbitrap analyzer were performed with on-the-fly internal recalibration (lock mass) at m/z 445.12002 (polydimethylcyclosiloxane).

2.5 Database searches Raw data files were processed using Proteome Discoverer 1.4 software (Thermo Scientific). Peak lists were searched using the MASCOT search software (Matrix Science) against the database

P.

aeruginosa

UCBPP-PA14

containing

5886

protein

sequences

(http://www.genoscope.cns.fr). Database searches were performed with the following parameters:

2

missed

trypsin

cleavage

sites

allowed;

variable

modifications:

carbamidomethylation on cysteine, oxidation on methionine, lysine succinylation (+100.0160) or lysine acetylation (+42.0105). The parent-ion and daughter-ion tolerances were 5 ppm and 6 ACS Paragon Plus Environment

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0.35 Da, respectively. False discovery rate (FDR) threshold for identifications was specified at 1% (for proteins and peptides). For each identification, we considered a peptide ion score higher than 15 for succinylation and 13 for acetylation; a peptide rank of 1, a q-value and an expectation value below 0.05; and a valid distribution of daughter ions (b- and y-fragment ion series) in the mass spectra, especially to certify the modified lysine. However, we didn't just sort according to these criteria, but we checked manually all spectra containing succinylation or acetylation to ensure the location of the PTM and the peptide sequence. Succinylated peptides with a good E-value but little information in their MS/MS spectra were removed. MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD008662.

2.6 Quantification To achieve a relative quantification of the modified peptides in each carbon source condition, we considered the nanoLC-MS/MS raw data after succinylated or acetylated peptide enrichment. To extract time-intensity chromatograms from MS1 scans, Skyline software (version 4.1.0.11796) was chosen using the MS1 Full-Scan Filtering method. The spectral library was built with the Proteome Discoverer results with a cut-off score of 0.95, and both lysine succinylation and acetylation were considered. All matching scans were included. The chromatogram peak areas of succinylated and acetylated peptides were extracted from the previous data dependent acquisition (DDA) for the 3 replicas per carbon source. To normalize, the chromatogram peak areas of each peptide were divided by the total chromatographic signal area of each sample.

2.7 Bioinformatics analysis of modified protein From the Pseudomonas genome database (www.pseudomonas.com), a lot of information about proteins were available and collected (gene name, protein name, localization, functional classifications and gene ontology (biological process, molecular function))54. The Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to annotate and to map the pathways. InterPro database were used to annotate protein domains. Protein-protein interactions were visualized with STRING software (version 9.1) with a high confidence score (0.7). Motif-X55 was used with default parameters to analyze sequence motifs (14 amino acids upstream and downstream of the succinylation site). Each hypothetical protein was BLAST on all Pseudomonas species on Pseudomonas genome database. If no match was found, BLAST was 7 ACS Paragon Plus Environment


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achieved on other bacteria species.

2.8 3D structure To identify the modified lysine positions on the proteins 3D structure, a model is built for each considered protein. First the protein sequence is blast on the sequences of proteins of known structure by choosing "protein data bank proteins (PDB)" database in the choose search set (https://blast.ncbi.nlm.nih.gov). Some of the analyzed proteins are already present in the PDB (http://www.rcsb.org/pdb/home/home.do). When it is not the case, but a high score is obtained (E value < 10-9, % identity > 30) for a large cover of the query sequence, a simple in silico mutation of the residues of interest is performed on the top five issuing proteins structures, which are superposed in PyMOL (http://www.pymol.org). When no structure with good confidence results from this analysis, a model is built by using different predicting sites (I-TASSER

(https://zhanglab.ccmb.med.umich.edu/I-TASSER/)

or

RaptorX

(http://raptorx.uchicago.edu/)) keeping all the parameters at their default values. The issued models are analyzed with caution based on their local structure accuracy profiles. The positions of modified amino acid are mapped on the modelled structures. The PyMOL program was used to visualize the structural results.


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3. Results and Discussion 3.1 Proteomics analyses of lysine succinylation and acetylation in different carbon sources in P. aeruginosa PA14 Lysine is a residue that can accept different types of chemical groups, as acetylation or succinylation. To establish the first repertory of lysine succinylation in P. aeruginosa strain PA14 and to compare it with that of lysine acetylation, we used a large-scale proteomic study (Figure 1). It is well known that the regulation of bacterial metabolism and PTM state are controlled by the carbon source. We consequently culture the bacterium in minimal medium enriched with different carbon sources, i.e., citrate, glucose, glutamate or succinate (Figure S1A in Supporting Information). After proteins digestion by trypsin, a direct analysis of the crude tryptic sample by nanoLC-MS/MS led first to the identification of less than 10 modified peptides. Modified peptides were obviously in a lower abundance than unmodified ones. Consequently, we decided to perform an enrichment of modified peptides. Despite the low reproducibility of immunoaffinity chromatography, it is currently the method of choice to trap modified peptides5. Succinylated and acetylated peptides were enriched by using a new twodimensional immunoaffinity chromatography56 (Figure 1). Succinylated peptides were first trapped with anti-succinylated lysine antibodies, and then analyzed by nanoLC-MS/MS. Peptides in the flow through were used for a second enrichment with anti-acetylated lysine antibodies. The selected peptides (acetylated) were analyzed by nanoLC-MS/MS. Peptides were identified from the Pseudomonas genome databank (www.pseudomonas.com) with a minimum of two missed cleavages since trypsin doesn’t cut after a modified lysine. By using this protocol, we were able, for the first time, to characterize succinylated and acetylated proteins in P. aeruginosa in the same sample and in 4 different carbon source (Table S1 in Supporting Information). The detailed information of the identified peptides and the matched proteins are listed in Table S2 and S3 in Supporting Information. Taken together, all the results allowed us to detect a total of 1 520 succinylated-lysine unique sites (1 522 peptides) occurring on 612 proteins. This corresponds to 10.5% of Open Reading Frames (ORF) in P. aeruginosa PA14. This percentage accords well with some previous investigations on γ-proteobacteria, e.g., Vibrio parahaemolyticus serotype O3:K638 or Escherichia coli MG165536 (Table S4 in Supporting Information). We also identified 1 102 acetylated-lysine unique sites (1 102 peptides) occurring on 522 proteins (i.e. 8.9% of ORFs). A previous study, on P. aeruginosa PA14 acetylome, achieved in glucose7, identified 320 acetylated proteins. We identified the same number of acetylated proteins (320 vs 341 proteins) but there is a low overlap of the acetylated proteins (24%) between the two studies 9 ACS Paragon Plus Environment


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likely due to low antibody reproductibility (see below). Carbon sources are known to influence PTMs rate. Thus, the number of succinylated proteins is greater in B. subtilis when cultured in the presence of citrate25. Conversely, a high concentration of glucose or succinate significantly increases the number of succinylated proteins in E. coli36,41. Finally, in C. glutamicum, an overproduction of glutamate leads to an increase in the number of succinylated proteins26. In accordance with these data, the carbon here altered the level of succinylated proteins. Citrate was the carbon source with which we observed the higher number of succinylated peptides (1 432 peptides) (Figure 2A) which represent 93.6% of the whole succinylated peptides that we identified in this investigation. In glucose, we identified the lower number of succinylated peptides. This was confirmed by Western Blot (Figure S1B in Supporting Information). It is also in citrate that we identified the higher number of acetylated peptides and in succinate that we observed the lower number for this modification (Figure 2B). However, in contrast with succinylation, the number of acetylated peptides was relatively homogeneous in the 4 carbon sources. Among the 4 carbon sources, citrate had a great impact on protein succinylation rate but a less impact on acetylation. An important drawback in immunoaffinity chromatography is the low specificity of antibodies, as previously noticed5, against succinylated or acetylated lysine peptides: only 20% of identified peptides were modified. However, this approach allows to characterize an in depth bacterial proteome. Indeed, the analyses of the crude sample (without enrichment) allow the identification of many unmodified proteins. Using the enrichment, we have access to proteins (15.4%) that were never identified in a crude sample, because probably to their low abundance.

3.2 Modified lysine sites We observe that the modified (succinylated and/or acetylated) lysine residues are distributed homogeneously on the protein sequence. Moreover, the number of modified lysine residues per protein is not proportional with the protein size. Whatever the PTMs (succinylation or acetylation) more than 50% of modified proteins had only one modified lysine sites and around 10% had more than 4 sites (Figure 2C). When considering both lysine succinylation and/or acetylation, the 2 most modified proteins are DnaK-PA14_62970 with 24 modified lysine and hypothetical protein-PA14_07680 (sharing 100% homology with serine protein kinase YeaG of P. fluorescence A506) with 22 modified lysines (Table S1 in Supporting Information). 10 ACS Paragon Plus Environment

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Among modified (succinylated and/or acetylated) proteins, 51 proteins (over 748 unique proteins) had 50% or more of their modified lysines (Table S1 in Supporting Information), like (i) phosphonate ABC transporter substrate-binding protein PhnD-PA14_20320 (14/28 modified lysines), (ii) GroEL-PA14_57010 (22/42 modified lysines), (iii) hypothetical protein-PA14_70680 sharing 100% homology with PstS of P. fluorescence F113 (15/28 modified lysines). Four proteins had 100% of their lysines modified: hypothetical proteinPA14_00900 (sharing 99.6% homology with Type VI secretion system protein FagF1 of P. aeruginosa PAO1), two hypothetical proteins-PA14_09780 and -PA14_38370 and chaperone GroES-PA14_57020. However, 3 of these 4 proteins possessed only one lysine in their sequence except GroES-PA14_57020 having 6 lysines, all modified by acetylation and/or succinylation.

3.3 Analysis of lysine succinylation motifs Different motifs surrounding succinylation sites were found in other bacterial species (Table S5 in Supporting Information). To determine if there are specific amino acids at position surrounding the succinylated lysine in PA14, we examined the amino acid sequence flanking modified sites based on Motif-X (p

Lysine succinylation and acetylation in Pseudomonas aeruginosa

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