Serine/threonine protein kinase PrkA of the human pathogen 2 Listeria monocytogenes : Biochemical characterization and 3 identification of interacting partners through 4 proteomic approaches

Listeria monocytogenes is the causative agent of listeriosis, a very serious food-borne human


Introduction
Listeria monocytogenes is a Gram positive rod-shaped bacterium that can be recovered from a wide range of sources such as soil, water, vegetation, effluents, human and animal feces and fresh and processed foods.This bacterium can tolerate hostile and stress conditions as high salt concentrations, acid pH and can grow at temperatures ranging from −1 °C to 45 °C [1].These features allow these bacteria to survive many of the strategies used for food preservation and thus they become an important threat for human health.As a result, L. monocytogenes arises as an important foodborn pathogen, etiologic agent of listeriosis, and a sporadic but very serious disease [2].Pregnant women, newborns, elderly and immunosuppressed individuals have predisposition to more severe presentation of the disease.In these high-risk populations, listeriosis can produce very serious clinical manifestations like septicemia, meningitis, meningoencephalitis and abortions, resulting in death in 20-30% of the cases despite early antibiotic treatment [1].Pathogenesis of L. monocytogenes is mediated by its ability to effectively invade and replicate within a broad range of eukaryotic cells and to cross the intestinal barrier, blood-brain barrier, and plancental barrier in the mammalian host.L. monocytogenes has a relatively complex infectious cycle with different stages: internalization in host cells, intracellular proliferation and intercellular spread.Each stage of the intracellular parasitism is dependent upon the differential expression of distinct virulence factors [3].
The extraordinary capacity of L. monocytogenes to adapt and respond to environmental changes seems to be related to an extensive repertoire of predicted regulatory proteins, including different RNA polymerase sigma factors, transcription factors and protein phosphorylation systems [4].Protein phosphorylation is a major mechanism in signal transduction processes by which environmental stimuli are translated into cellular responses and represents one of the most important post-translational modifications regulating enzyme activities and protein interactions [5,6].Signal transduction in prokaryotes is predominantly accomplished by the so called two-component systems, consisting of His-kinase sensors and their associated response regulators [7].In contrast, in eukaryotes such signaling pathways are mainly carried out by Ser/Thr or Tyr-kinases [8].Long time thought to be exclusive to eukaryotes, a bulk of evidence raised from genome sequence data now indicates that Ser, Thr, and Tyr phosphorylation is also widespread in prokaryotes [9].These eukaryotic-like signaling systems have been shown to control essential processes in bacteria, including development, cell growth, stress responses, central and secondary metabolism, biofilm formation, antibiotic resistance, and virulence [9][10][11][12][13][14][15].In the case of L. monocytogenes the presence of eukaryotic-like phosphorylation systems has been predicted by genome analysis.In particular, it was reported that the stp gene (lmo1821) encodes a functional Ser/Thr protein phosphatase (STPP) required for growth of L. monocytogenes and virulence in murine model of infection.In addition, the elongation factor EF-Tu was described as a target for this phosphatase [16].
However, there is no information regarding the corresponding phosphorylating enzymes, endogenous substrates and their role in bacteria physiology and physiopathology.
In the present work we report the cloning, expression and purification of the catalytic domain of the gene product of The gels were silver stained according to protocols described [22].Images were digitalized using a UMAX Power-Look 1120 scanner and LabScan 5.0 software (GE Healthcare).

Results and discussion
3.1.

Sequence analysis
The The STPK PrkA is a predicted 655 amino acids transmembrane protein, with a theoretical molecular mass of 72 kDa and a pI value of 4.99.Sequence analysis showed the presence of a pattern of basic residues followed by a predicted transmembrane domain suggesting that the N-terminal region (residues 1-338) is orientated toward the cytoplasm [30].It was also observed that PrkA N-terminal sequence contains a predicted STPK that exhibits all the conserved subdomanis (subdomains I to V, VIa, VIb and VII to XI) and the nearly invariant residues that define the Hanks family of eukaryotic protein kinases [8] (Fig. 2 additional bands ranging from 41 to 43 kDa (Fig. 3).All these To test if phosphorylation of the activation loop sequence was a result of an autocatalytic reaction, the recombinant kinase was de-phosphorylated using alkaline phosphatase, purified using Ni 2+ -NTA resin and re-incubated in the presence of ATP and Mn 2+ .The phosphorylation status of PrkAc was followed by Organization of the genome region enclosing the gene that encodes for the putative Ser/Thr protein kinase PrkA.Arrows indicate the orientation of transcription.This region encodes six ORFs involved in information pathways (dark gray) and two ORFs involved in secondary metabolism (light gray).lmo1818: similar to ribulose-5-phosphate 3-epimerase; lmo1819: similar to ribosome associated GTPase; lmo1820: PrkA, similar to putative Ser/Thr-specific protein kinase; lmo1821: similar to putative phosphoprotein phosphatase; lmo1822: similar to RNA-binding Sun protein; fmt: similar to methionyl-tRNA formyltransferase; priA: similar to primosomal replication factor Y; lmo1825: similar to pantothenate metabolism flavoprotein homolog; STPK: Ser/ Thr protein kinase; STPP pSer/pThr protein phosphatase.
MS analysis after proteolytic treatment.Spectra analysis showed 468 that phosphatase treatment results in activation loop de- The activation loop phosphorylation status is important to control the active/inactive conformational switch in numerous kinases.A wide range of regulatory mechanism has been suggested for this loop, such as the contribution to the appropriate alignment of the catalytic residues and the correction of the relative orientation of different domains allowing the binding of the protein substrate and/or ATP [42].
The relevance of the activation loop phosphorylation has been but also defines a high affinity docking site that is relevant for substrate recruitment [43].Considering these evidences from homologous proteins, we can suggest that the very well conserved phosphorylation pattern here reported for PrkA, participates in activity control and perhaps also in substrate recruitment by protein interactions mediated by specific phospho-residues recognition.

Identification of putative interacting partners of PrkAc
As a first approach to reveal possible interactions between phosphorylated PrkAc and proteins from L. monocytogenes cellular extracts, we used a surface plasmon resonance strategy.
These experiments allowed us to determine that immobilized PrkAc interacted with components of L. monocytogenes protein extract (data not shown).
In order to identify the proteins that possibly interact with  independent experiments that clearly differed from the 2D 528 profile of total cellular extracts (data not shown).Spots 529 detected in all replicates were processed for protein identifi-530 cation by PMF (Fig. 7 and supplementary Fig. 2).This strategy of carbohydrates (26%) and protein synthesis (19%) (Fig. 8).

541
This is followed by proteins involved in transport and binding 542 of proteins and lipoproteins (10%) and in cell wall metabolism 543 (9%).A primary conclusion that arises from the diversity of 544 proteins identified as potential interaction partners of PrkAc could be that the signal transduction pathways mediated by this STPK in L. monocytogenes could be affecting a great variety of fundamental biological functions.
Since the immobilized protein is the autophosphorylated catalytic domain of a STPK, we consider the possibility that some of the potential interacting partners were also substrates of the kinase.Therefore we searched reported phosphoproteomes to see if the identified proteins were phosphorylated at Ser or Thr in other microorganism.We found that 48% of the proteins were described to be phosphorylated in at least one of the following microorganisms: C. glutamicum, B. subtilis, E. coli, M. tuberculosis, Pseudomonas aeruginosa, P. putida, Lactococcus lactis, S. pneumoniae, and Campylobacter jejuni [44][45][46][47][48][49][50][51][52][53].
It is also important to note that many of these putative partners were reported as the proteins most frequently identified in differential expression proteomic analysis based on 2D gel approaches [54,55].If the identification of these proteins represents a technical artifact or reveals that they participate in a general cell mechanism is still a matter of debate [54,55].Even when our experimental approach points to a specific interaction of these proteins with PrkAc, we have to be very careful with the interpretation of these results.In addition to these frequently detected proteins, less abundant regulatory proteins were also identified as possible interactors of PrkAc.
The list of proteins and protein families identified provides information regarding possible functions of PrkAc.In the following paragraphs we focus on some of the potential interaction partners of PrkAc that are related to STPKs function in other organisms and whose relevance has been reported or strongly suggested.

Proteins involved in the carbohydrate metabolism
We identified 15 proteins related to the glycolytic pathway and the tricarboxylic acid (TCA) cycle.Some of them (aldolase, glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase, lactate dehydrogenase, acetate kinase, dihydrolipoamide dehydrogenase and α-cetoglutarate dehydrogenase) were found to be phosphorylated at Ser, Thr or Tyr residues trough phosphoprotemic studies in other microorganisms [44][45][46][47][48][49][50][51][52][53].It was also proved that the transcriptional profile of two enzymes involved in the TCA cycle (dihydrolipoamide succinyltransferase and oxoglutarate dehydrogenase E1) is affected by the STPK PknB from S. aureus [56].Additionally, in M. tuberculosis and C. glutamicum it has been demonstrated that the regulation of TCA cycle is mediated by STPKs [57,58].In these bacteria, the STPKs PknB and PknG phosphorylate a protein containing a FHA domain (GarA y OdhI in M. tuberculosis and C. glutamicum respectively) which in their de-phosphorylated forms inhibit the enzyme 2-oxoglutarato dehydrogenase [57,58].FHA domains are small protein modules that mediate protein-protein interactions in the STPK-mediated signal transduction pathways through the recognition of specific phosphorylated residues [59].
Genome sequence analyses have revealed that all members of the order actinomycetales present GarA-homologous proteins which show strong sequence conservation at the C-terminus FHA domain [43].However, the analysis of the proteins coded by the L. monocytogenes genome does not predict the presence FHAcontaining proteins.Therefore, the STPK PrkA in L. monocytogenes could be involved in the modulation of the TCA cycle through a Please cite this article as: Lima analysis of the L. monocytogenes EGDe genome revealed the presence of two putative STPKs (lmo0618 and lmo1820) and one STPP (lmo1821).In the 10.2 kbp region that encloses the gene coding PrkA (lmo1820) eight open reading frames are found (http://genolist.pasteur.fr/ListiList/)(Fig. 1).This gene cluster also includes the gene lmo1821 and other genes involved in information pathways (DNA, RNA and protein metabolism and modification) (lmo1819, lmo1822, fmt, and priA) and intermediary metabolism (lmo1818 and lmo1825).The presence in the same genome region of a STPP gene preceding the STPK gene was also found in other bacteria suggesting a functional association between theses enzymes [23-27].Particularly it has been observed that such STPK/STPP couples act as functional pairs in Mycobacterium tuberculosis, Staphylococcus aureus and Bacillus subtilis [23,25,28,29].

390
proteins were identified by PMF as PrkA demonstrating that the 391 protein expressed in E. coli has at least three isoforms with 392 different migration behavior in SDS-PAGE.393Therecombinant protein PrkAc was examined for its ability 394 to phosphorylate the exogenous substrate MBP.Comparison 395 of mass spectra of digested MBP after and before incubation 396 with PrkAc in the presence of ATP and Mn 2 revealed that 397 sequence 30-41 is phosphorylated by the kinase.Signal of 398 native sequence (m/z = 1339.61)present in control spectra 399 decreased after phosphorylation reaction and concomitantly 400 a signal with a mass increment of 80 Da (m/z = 1419.68)became 401 apparent (Fig.4).This particular MBP peptide was found to be 402 systematically and extensively phosphorylated by several myco-403 bacterial STPKs.Its detection by MS was previously reported as a 404 sensitive marker of kinase activity[36].Phosphorylation of MBP 405 tryptic peptide 30-41 by PrkAc was further confirmed by MS/MS 406 analysis (Fig.4).The presence of daughter ions with mass 407 differences of 80 Da (loss of HPO 3 ) and 98 Da (loss of H 3 PO 4 ) is 408 characteristic of phosphorylated peptides[36,37].These results409clearly demonstrate that PrkAc was produced in E. coli as a 410 functional STPK able to phosphorylate the exogenous substrate 411 MBP.The fact that PrkAc phosphorylates the same MBP peptide 412 than mycobacterial protein kinases probably reflects some 413 specificity of bacterial kinases towards this sequence.414 3.3.Identification of phosphorylated peptides and residues 415 in PrkAc 416 The overall phosphorylation status of the recombinant kinase 417 was tested by MALDI-TOF mass measurements of tryptic 418 digestions of PrkAc before and after the treatment with alkaline 419 phosphatase.Results obtained from spectra comparison allowed 420 us to predict the presence of phospho-Ser and phospho-Thr containing peptides (m/z=3733.72,m/z=3813.96,and m/z= 3893.90 could be assigned to the mono-, di-, and tri-phosphorylated tryptic peptide 160-183 respectively) (Fig. 5).Additionally, the multiple phosphorylated state of these peptides was confirmed by MS/MS analyses (data not shown).It is interesting to note that this multiple phosphorylated peptide is enclosed within the conserved motifs DFG and PE of Hanks kinases corresponding to the activation loop in several STPK from related bacteria [8,23,25,36,38-40].The identification of phosphorylation sites by MS/MS analyses is usually challenging because fragmentation of phosphopeptides is mainly dominated by the neutral loss of phosphate group.This fact precludes the detection of sequence-specific ion signals rendering difficult the localization of modification sites [36].For that reason, we treated the phosphorylated peptides with Ba(OH) 2 to generate de-hydro amino acids from phospho-Ser and phospho-Thr residues by β-elimination of H 3 PO 4 .Such derivatives have better properties for MS/MS experiments.Moreover they show a mass difference of 18 Da compared to the parent amino acid residue, thus becoming a useful tag for phosphoresidue identification [41].The spectrum of Ba(OH) 2 treated peptides showed signals 18, 36 and 54 Da lower than the expected for native peptides 160-183, indicating the presence of species that have been generated by multiple β-elimination of phosphate group (Fig. 6).The phosphorylation sites were assigned by manual inspection of MS/MS spectrum of the ion generated after β-elimination reaction of the tri-phosphorylated peptide.This spectrum shows mostly y-ions and the presence of signals with mass differences of 18 Da (and multiple thereof) in relation to the theoretical expected values, was clearly detected allowing the unequivocally identification of modified residues (Fig. 6).The results allowed us to identify the phosphorylation sites as Thr171, Thr174 and Thr176 within the sequence 160-183 of PrkA activation loop.At least two of this Thr residues are highly conserved in the activation loop sequence of other bacterial STPKs and its phosphorylated state has been reported [23,35,36,38,40].In addition, it was demonstrated for some STPKs, such as PrkC from B. subtilis and PknB from M. tuberculosis, that the phosphorylation of these conserved Thr residues in the activation loop regulates kinase activity [23,35].

Fig. 1 -
Fig. 1 -Q1Organization of the genome region enclosing the gene that encodes for the putative Ser/Thr protein kinase PrkA.Arrows indicate the orientation of transcription.This region encodes six ORFs involved in information pathways (dark gray) and two ORFs involved in secondary metabolism (light gray).lmo1818: similar to ribulose-5-phosphate 3-epimerase; lmo1819: similar to ribosome associated GTPase; lmo1820: PrkA, similar to putative Ser/Thr-specific protein kinase; lmo1821: similar to putative phosphoprotein phosphatase; lmo1822: similar to RNA-binding Sun protein; fmt: similar to methionyl-tRNA formyltransferase; priA: similar to primosomal replication factor Y; lmo1825: similar to pantothenate metabolism flavoprotein homolog; STPK: Ser/ Thr protein kinase; STPP pSer/pThr protein phosphatase.
469 phosphorylation, indicated by the disappearance of phosphor-470 ylated species and the increase of native peptide m/z signal.After 471 incubation of the de-phosphorylated enzyme with ATP the 472 activation loop phosphopeptides were clearly detected in the 473 mass spectrum, indicating that PrkAc presented autocatalytic 474 activity (data not shown).

Fig. 2 - 6 J
Fig. 2 -Protein sequence alignment of the N-terminal domain of PrkA and catalytic domains of other characterized bacterial Ser/ Thr protein kinases.PrkA, putative STPK from L. monocytogenes; PrkC, from B. subtilis; Stk, from S. aureus; StkP, from S. pneumoniae; and PknB, from M. tuberculosis.Sequences alignment was performed with ClustalW and GeneDoc softwares.Sequences showing 100% of conservation are shaded in black (identical residues and conservative changes).Sequences showing more than 60% and 40% of conservation are indicated in dark and light gray respectively.Sub-domains I-IX that define the Hanks family of eukaryotic-like protein kinases are indicated above and nearly invariant residues are indicated below the alignment.
PrkAc we carried out affinity chromatography experiments using the conditions obtained from surface plasmon resonance experiments.For that purposes, we first immobilized recombinant PrkAc to a Hi-trap NHS-activated resin HP (Amersham Bioscience).A fraction of the resin submitted to the process of immobilization was digested with trypsin and analyzed by MS to confirm the coupling of PrkAc.Only tryptic masses from PrkAc were detected, discarding the presence of significant amounts of contaminating proteins.The incubation of the covalently bound kinase with MBP under phosphorylation conditions showed that the immobilized protein was an active enzyme (data not shown).To recover either individual proteins or protein complexes that bind to PrkAc, we incubated the modified and control 516 resin with a soluble protein extract from L. monocytogenes 517 EGDe.After extensive washing the ligands were eluted using 518 acid pH.The different fractions of the affinity chromatography 519 were primarily analyzed by one-dimensional SDS-PAGE and 520 visualized by silver staining.From these analyses we could 521 observed that many proteins were retained by PrkAc resin 522 while we did not detect proteins in control resins (data not 523 shown).524 In order to achieve a better resolution, eluted protein were 525 separated by 2D electrophoresis.Analysis of 2D gels allowed 526 us to detect a specific protein profile of eluted proteins in

Fig. 3 -Fig. 4 -
Fig. 3 -Over-expression and purification of His 6 -tagged PrkAc.Proteins were purified with Ni 2+ -NTA resin, separated on 12.5% SDS-PAGE and stained with Coomassie blue.Lane 1: molecular weight marker (Amersham Low Molecular Weight Calibration Kit for SDS Electrophoresis); lanes 2-5: different fractions eluted with 500 mM imizadol.At least 3 bands ranging from 39 to 43 kDa were detected in the eluted fractions and were identified as PrkA from L. monocytogenes by PMF.

Fig. 5 -
Fig. 5 -Detection of phosporylated peptides in PrkAc.Mass spectra of tryptic digestion of PrkAc before (A) and after (B) the treatment with alkaline phosphatase.Mass signals corresponding to native peptide 150-183 (MH + ) and its mono-, di-and tri-phoshorylated ions, showing a mass shift in 80 Da and multiples thereof, are indicated with arrows.The multiple phosphorylation of the sequences 150-183 was confirmed by the disappearance of the corresponding ions from the spectrum after phosphatase treatment.

Fig. 6 - 9 J
Fig. 6 -Identification of phosphorylation sites by MS/MS analysis.(A) Spectrum of tryptic digestion of PrkAc after treatment with Ba(OH) 2 .The appearance of mass signals differing in 18 Da, 36 Da and 54 Da from native peptide 160-183 confirmed the β-elimination of one, two and three phosphate groups respectively.(B) MS/MS analysis of peptide generated from tri-phosphorylated species after β-elimination reaction.The occurrence of y-ions with mass difference of 18 Da (and multiples) allowed the identification of de-hydrated Ser and Thr residues generated from previously phosphorylated residues by β-elimination reaction.(C) 160-183 sequence showing the identified modified residues.

664 3 . 4 . 5 .
Proteins involved in adaptation to atypical conditions 665 and detoxification 666 The protein similar to ATP-dependent Clp protease proteolytic 667 component, classified as a protein implicated in the adapta-668 tion to atypical conditions, and the proteins involved in 669 detoxification, superoxide dismutase and thiol peroxidase 670 were identified as putative interactors of PrkAc.All of these 671proteins were found phosphorylated in other organisms 672[48,51].Particularly, it was reported that the activity of the superoxide dismutase from L. monocytogenes is regulated by phosphorylation at Ser and Thr residues being most active at its non-phosphorylated form[70].In summary, in the present work we identify 62 candidates that provide a starting point for further biochemical and cellular studies.The physiological relevance of the proteins and protein families identified in this interactome analysis has to be further examined.According to recent proteomic meta-analysis many of these proteins families (including glycolytic enzymes and elongation factors) are frequently detected as differentially expressed in various conditions raising concern about their specificity[54,55].Based on previous reports we can hypothesize that some of these frequently identified proteins present in t1:62

Table 1
t1:1 -Proteins identified as putative interaction partners of PrkAc classified according to their functional category a .
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