Eric Martinez

Sylvaine Huc-Brandt

Solène Brelle

Julie Allombert

Franck Cantet

Laila Gannoun-Zaki

Mélanie Burette

Marianne Martin

François Letourneur

Matteo Bonazzi
email: matteo.bonazzi@irim.cnrs.fr

Virginie Molle
email: virginie.molle@umontpellier.fr

The secreted protein kinase CstK from Coxiella burnetii influences vacuole development and interacts with the GTPase-activating host protein TBC1D5

Keywords: burnetii vacuole biogenesis Coxiella burnetii, secreted kinase, phosphorylation, serine/threonine/tyrosine protein kinase, parasitophorous vacuole, host substrates, Q fever, bacterial effector, virulence factor

The intracellular bacterial pathogen Coxiella burnetii is the etiological agent of the emerging zoonosis Q fever. Crucial to its pathogenesis is type 4b secretion system (T4SS)-mediated secretion of bacterial effectors into host cells that subvert host cell membrane trafficking, leading to the biogenesis of a parasitophorous vacuole for intracellular replication. The characterization of prokaryotic Serine/Threonine Protein Kinases (STPKs) in bacterial pathogens is emerging as an important strategy to better understand hostpathogen interactions. In this study, we investigated CstK (for Coxiella ser/thr Kinase), a protein kinase identified in C. burnetii by in silico analysis. We demonstrate that this putative protein kinase undergoes autophosphorylation on Thr and Tyr residues, and phosphorylates a classical eukaryotic protein kinase substrate in vitro. This dual Thr-Tyr kinase activity is also observed for a eukaryotic dual-specificity Tyr phosphorylationregulated kinase class. We found that CstK is translocated during infections and localizes to Coxiella-containing vacuoles (CCVs). Moreover, a CstK-overexpressing C. burnetii strain displayed a severe CCV development phenotype, suggesting that CstK fine-tunes CCV biogenesis during the infection. Protein-protein interaction experiments identified the Rab7 GTPase-activating protein (GAP) TBC1D5 as a candidate CstK-specific target, suggesting a role for this host GAP in Coxiella infections. Indeed, CstK colocalized with TBC1D5 in non-infected cells, and TBC1D5 was recruited to CCVs in infected cells. Accordingly, TBC1D5 depletion from infected cells significantly affected CCV development. Our results indicate that CstK functions as a bacterial effector protein that interacts with the host protein TBC1D5 during vacuole biogenesis and intracellular replication.

Signal transduction is an essential and universal function that allows all cells, from prokaryotes to eukaryotes, to translate environmental signals to adaptive changes. By this mechanism, extracellular inputs propagate through complex signaling networks whose activity is often regulated by reversible protein phosphorylation. Signaling mediated by Serine/Threonine/Tyrosine protein phosphorylation has been extensively studied in eukaryotes, however, its relevance in prokaryotes has only begun to be appreciated. The recent discovery that bacteria also use Ser/Thr/Tyr kinase-based signaling pathways has opened new perspectives to study environmental adaptation, especially in the case of bacterial pathogens, with respect to host infection [START_REF] Stancik | Serine/Threonine Protein Kinases from Bacteria, Archaea and Eukarya Share a Common Evolutionary Origin Deeply Rooted in the Tree of Life[END_REF]. Thus, advances in genetic strategies and genome sequencing have revealed the existence of "eukaryotic-like" serine/threonine protein kinases (STPKs) and phosphatases in a number of prokaryotic organisms [START_REF] Janczarek | Hanks-Type Serine/Threonine Protein Kinases and Phosphatases in Bacteria: Roles in Signaling and Adaptation to Various Environments[END_REF], including pathogens such as, Streptococcus spp. [START_REF] Rajagopal | A eukaryotic type serine/threonine kinase and phosphatase in Streptococcus agalactiae reversibly phosphorylate an inorganic pyrophosphatase and affect growth, cell segregation, and virulence[END_REF][START_REF] Hussain | A eukaryotic-type serine/threonine protein kinase is required for biofilm formation, genetic competence, and acid resistance in Streptococcus mutans[END_REF][START_REF] Echenique | Protein serine/threonine kinase StkP positively controls virulence and competence in Streptococcus pneumoniae[END_REF][START_REF] Jin | Identification and biochemical characterization of a eukaryotictype serine/threonine kinase and its cognate phosphatase in Streptococcus pyogenes: their biological functions and substrate identification[END_REF], Mycobacteria [START_REF] Av-Gay | The eukaryotic-like Ser/Thr protein kinases of Mycobacterium tuberculosis[END_REF][START_REF] Chaba | Evidence that a eukaryotic-type serine/threonine protein kinase from Mycobacterium tuberculosis regulates morphological changes associated with cell division[END_REF][START_REF] Wehenkel | Mycobacterial Ser/Thr protein kinases and phosphatases: physiological roles and therapeutic potential[END_REF][START_REF] Molle | Division and cell envelope regulation by Ser/Thr phosphorylation: Mycobacterium shows the way[END_REF][START_REF] Alber | Signaling mechanisms of the Mycobacterium tuberculosis receptor Ser/Thr protein kinases[END_REF][START_REF] Greenstein | Structure/function studies of Ser/Thr and Tyr protein phosphorylation in Mycobacterium tuberculosis[END_REF], Yersinia spp. [START_REF] Galyov | A secreted protein kinase of Yersinia pseudotuberculosis is an indispensable virulence determinant[END_REF][START_REF] Hakansson | The Yersinia YpkA Ser/Thr kinase is translocated and subsequently targeted to the inner surface of the HeLa cell plasma membrane[END_REF], Listeria monocytogenes [START_REF] Archambaud | Translation elongation factor EF-Tu is a target for Stp, a serine-threonine phosphatase involved in virulence of Listeria monocytogenes[END_REF][START_REF] Lima | Serine/threonine protein kinase PrkA of the human pathogen Listeria monocytogenes: biochemical characterization and identification of interacting partners through proteomic approaches[END_REF], Pseudomonas aeruginosa [START_REF] Wang | A novel serine/threonine protein kinase homologue of Pseudomonas aeruginosa is specifically inducible within the host infection site and is required for full virulence in neutropenic mice[END_REF], Enterococcus faecalis [START_REF] Kristich | A eukaryotic-type Ser/Thr kinase in Enterococcus faecalis mediates antimicrobial resistance and intestinal persistence[END_REF] or Staphylococcus aureus [START_REF] Beltramini | Modulation of cell wall structure and antimicrobial susceptibility by a Staphylococcus aureus eukaryote-like serine/threonine kinase and phosphatase[END_REF][START_REF] Truong-Bolduc | Posttranslational modification influences the effects of MgrA on norA expression in Staphylococcus aureus[END_REF]. Consequently, the study of STPKs in human bacterial pathogens is emerging as an important strategy to better understand host-pathogen interactions and develop new, targeted antimicrobial therapies. However, if on one hand it is clear that STPKs and phosphatases regulate important functions in bacterial pathogens, their signal transduction mechanism remains ill-defined and restricted to a limited number of microbes.

Importantly, STPKs expressed by pathogenic bacteria can either act as key regulators of important microbial processes, or be translocated by secretion systems to interact with host substrates, thereby subverting essential host functions including the immune response, cell shape and integrity [START_REF] Canova | Bacterial serine/threonine protein kinases in host-pathogen interactions[END_REF]. Phosphorylation of host substrates has been demonstrated for some bacterial STPKs, whereas others seem to require their kinase activity but their phosphorylated substrates remain to be identified [START_REF] Canova | Bacterial serine/threonine protein kinases in host-pathogen interactions[END_REF]. Therefore, biochemical mechanisms of these pathogendirected targeted perturbations in the host cellsignaling network are being actively investigated and STPKs are proving to be molecular switches that play key roles in host-pathogen interactions [START_REF] Canova | Bacterial serine/threonine protein kinases in host-pathogen interactions[END_REF].

Among emerging human pathogens, Coxiella burnetii is a highly infectious bacterium, responsible for the zoonosis Q fever, a debilitating flu-like disease leading to large outbreaks with a severe health and economic burden [START_REF] Kazar | Coxiella burnetii infection[END_REF][START_REF] Arricau-Bouvery | Is Q fever an emerging or re-emerging zoonosis?[END_REF][START_REF] Madariaga | Q fever: a biological weapon in your backyard[END_REF]. The efficiency of infections by C. burnetii is likely associated with the remarkable capacity of this bacterium to adapt to environmental as well as intracellular stress. Indeed, outside the host, C. burnetii generates pseudo-spores that facilitate its airborne dissemination. C. burnetii has developed a unique adaptation to the host, being the only bacterium that thrives in an acidic compartment containing active lysosomal enzymes. Upon host cell invasion, bacteria reside within membrane-bound compartments that passively traffic through the endocytic maturation pathway, progressively acquiring early and late endocytic markers such as Rab5 and Rab7, respectively [START_REF] Beron | Coxiella burnetii localizes in a Rab7-labeled compartment with autophagic characteristics[END_REF]. Fusion of Coxiella-containing vacuoles (CCVs) with late endosomes and lysosomes is accompanied by the acidification of the endosomal environment, which is required to activate the translocation of bacterial effector proteins by a Dot/Icm Type 4b Secretion System (T4SS) [START_REF] Newton | Effector protein translocation by the Coxiella burnetii Dot/Icm type IV secretion system requires endocytic maturation of the pathogenoccupied vacuole[END_REF]. Some of these effectors modulate important signaling pathways of infected cells, including apoptosis and inflammasome activation [START_REF] Luhrmann | Inhibition of pathogen-induced apoptosis by a Coxiella burnetii type IV effector protein[END_REF][START_REF] Klingenbeck | The Coxiella burnetii type IV secretion system substrate CaeB inhibits intrinsic apoptosis at the mitochondrial level[END_REF][START_REF] Cunha | Inhibition of inflammasome activation by Coxiella burnetii type IV secretion system effector IcaA[END_REF], whereas others are essential for the development of the intracellular replicative niche. Among these CvpB and CvpF have been recently implicated in the manipulation of autophagy for optimal vacuole development [START_REF] Martinez | Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development[END_REF][START_REF] Siadous | Coxiella effector protein CvpF subverts RAB26-dependent autophagy to promote vacuole biogenesis and virulence[END_REF](32)[START_REF] Latomanski | The Effector Cig57 Hijacks FCHO-Mediated Vesicular Trafficking to Facilitate Intracellular Replication of Coxiella burnetii[END_REF]. C. burnetii genome analysis revealed a close homology to the facultative intracellular pathogen Legionella pneumophila, in particular at the level of Dot/Icm core genes [START_REF] Vogel | Turning a tiger into a house cat: using Legionella pneumophila to study Coxiella burnetii[END_REF]. In silico analysis identified over 100 candidate effector proteins encoded in the C. burnetii genome, some of which have been validated for secretion using either C. burnetii or L. pneumophila as a surrogate model [START_REF] Newton | Effector protein translocation by the Coxiella burnetii Dot/Icm type IV secretion system requires endocytic maturation of the pathogenoccupied vacuole[END_REF][START_REF] Carey | The Coxiella burnetii Dot/Icm system delivers a unique repertoire of type IV effectors into host cells and is required for intracellular replication[END_REF][START_REF] Chen | Large-scale identification and translocation of type IV secretion substrates by Coxiella burnetii[END_REF]. In this study, we investigated the candidate effector CBU_0175, which encodes a unique putative Coxiella Ser/Thr kinase (CstK). We demonstrated CstK translocation by C. burnetii during infection and we reported its localization at CCVs. In vitro kinase assays revealed that CstK undergoes autophosphorylation on Thr and Tyr residues, and displays a 'bona fide' kinase activity towards a test substrate of eukaryotic protein kinases. Furthermore, the identification of the Rab7 GTPactivating protein (GAP) TBC1D5 as a CstK interactor suggests that this protein might be involved during infection to facilitate CCVs biogenesis. Indeed, TBC1D5 is actively recruited at CCVs during Coxiella infections and TBC1D5targeting siRNAs significantly affect CCVs development. Our data provide the first evidence that a C. burnetii secreted kinase might control host cell infection.

RESULTS

Coxiella burnetii genome encodes a single putative protein kinase.

In silico analysis of the virulent C. burnetii strain RSA493 NMI genome revealed only one gene encoding a putative Serine/Threonine Protein Kinase (STPK). To date, no STPKs have been characterized in this organism. This gene was named cstK for C. burnetii serine threonine Kinase and encodes a 246 amino acids protein with an estimated molecular mass of 31 kDa. The gene coding for cstK is flanked by genes CBU_0174 (which encodes an hypothetical protein) and CBU_0176, a gene coding for the serine protease domain-containing protein degP.1. Of note, these genes are not part of an operon (Fig. 1A). InterProScan analysis of CstK revealed the presence of most of the essential amino acids and sequence subdomains characterizing the Hanks family of eukaryotic-like protein kinases [START_REF] Hanks | Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification[END_REF]. CstK shares a common eukaryotic protein kinases superfamily fold with two lobes and a Gly-rich loop. These include the central core of the catalytic domain, and the invariant lysine residue in the consensus motif within subdomain II, which is usually involved in the phosphate transfer reaction and required for the autophosphorylating activity of eukaryotic STPKs (Fig. 1A) [START_REF] Hanks | Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification[END_REF][START_REF] Hanks | The protein kinase family: conserved features and deduced phylogeny of the catalytic domains[END_REF][START_REF] Carrera | The conserved lysine of the catalytic domain of protein kinases is actively involved in the phosphotransfer reaction and not required for anchoring ATP[END_REF]. The activation loop in the catalytic domain is particularly short in CstK, and the DFG motif is substituted by a GLG motif. Interestingly, the transmembrane domain usually present in classical prokaryotic STPKs is lacking in CstK, thus it is a so-called cytoplasmic STPK.

CstK is a Dot/Icm effector protein. Bioinformatics analysis using the prediction software S4TE 2.0 [START_REF] Noroy | Searching Algorithm for Type IV Effector proteins (S4TE) 2.0: improved tools for type IV effector prediction, analysis and comparison[END_REF] indicated that CstK harbors features corresponding to secreted effector proteins, including a promoter motif typically found in effector proteins from intra-vacuolar bacterial pathogens, suggesting that CstK is indeed a Coxiella effector protein (Fig. 1A). Consistently, previous studies by Chen and colleagues have shown that CstK is secreted in a T4SS-dependent manner by the surrogate host L. pneumophila, albeit with low efficiency [START_REF] Chen | Large-scale identification and translocation of type IV secretion substrates by Coxiella burnetii[END_REF]. In order to validate CstK secretion in C. burnetii, we engineered plasmids encoding, either CstK or CvpB (a known C. burnetii effector protein) [START_REF] Martinez | Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development[END_REF], fused to Beta-Lactamase (BLAM) and expressed in wt Coxiella Tn1832 (a C. burnetii transposon mutant expressing GFP and that phenocopies wild-type C. burnetii) or the Dot/Icm-defective dotA::Tn mutant, also expressing GFP. By means of a BLAM secretion assay, we could observe that BLAM-CstK was secreted by wt Coxiella at 48 and 72 hours, but not at 24 hours post-infection (Fig. 1B). Secretion of BLAM-CvpB or BLAM-CstK was not detectable in cells infected with the dotA::Tn strain, indicating that both CvpB and CstK are C. burnetii Dot/Icm substrates (Fig. 1B). Next, the intracellular localization of Cstk was investigated by ectopically expressing HA-tagged CstK either in non-infected or wt C. burnetiiinfected U2OS cells. In non-infected cells, CstK localized at intracellular compartments that were negative for the lysosomal marker LAMP1, whereas it was recruited at CCVs (as revealed by the co-localization with LAMP1) in infected cells (Fig. 1C).

CstK displays autokinase and protein kinase activities.

To determine whether CstK is a functional protein kinase, this protein was overproduced in E. coli and purified as a recombinant protein fused to glutathione Stransferase (GST) tag. The purified tagged CstK protein (Fig. 2A, upper panel) was then assayed for autokinase activity in presence of the phosphate donor [γ-33 P]ATP. As shown in Fig. 2A (lower panel), CstK incorporated radioactive phosphate from [γ-33 P]ATP, generating a radioactive signal corresponding to the expected size of the protein isoform, strongly suggesting that this kinase undergoes autophosphorylation. To confirm CstK autophosphorylation and exclude that contaminant kinase activities from E. coli extracts might phosphorylate CstK, we mutated the conserved Lys55 residue present in subdomain II into CstK by site-directed mutagenesis. Indeed protein sequence analysis revealed that Lys55 in CstK is similarly positioned as a conserved Lys residue usually involved in the phosphotransfer reaction and also required for the autophosphorylating activity of eukaryotic-like STPKs [START_REF] Hanks | Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification[END_REF][START_REF] Hanks | The protein kinase family: conserved features and deduced phylogeny of the catalytic domains[END_REF]. Thus, Lys55 was substituted by a Met residue, the mutated form of CstK, CstK_K55M, was purified as described above (Fig. 2A, upper panel) and it was then tested for autophosphorylation in presence of [γ-33 P]ATP. As expected, no radioactive signal could be detected (Fig. 2A, lower panel), thus establishing that CstK displayed autophosphorylation activity. A kinetic analysis of CstK phosphorylation was next performed to determine the initial CstK phosphorylation rate (Fig. 2B). Incorporation of γ-phosphate occurred rapidly, reaching about 50% of its maximum rate within 5 min of reaction. This autokinase activity was dependent on bivalent cations such as Mg 2+ and Mn 2+ in the range of 5mM, thus in correlation with concentrations required for canonical STPK activity, as shown in Fig. 2C, and abolished by addition of 20 mM EDTA chelating all the divalent cations available (data not shown).

The recombinant CstK protein was further characterized by studying its ability to phosphorylate exogenous proteins and was thus assayed for in vitro phosphorylation of the general eukaryotic protein kinase substrate, myelin basic protein (MBP), in the presence of [γ-33 P]ATP. MBP is a commonly used substrate for both Ser/Thr and Tyr kinases. A radiolabeled signal at the expected 18-kDa molecular mass of MBP was detected, thus demonstrating that CstK phosphorylates protein substrates such as MBP (Fig. 2A). As expected, the CstK_K55M mutant did not phosphorylated MBP. Altogether, these data indicate that in vitro, CstK possesses intrinsic autophosphorylation activity and displays kinase functions for exogenous substrates.

Identification of CstK autophosphorylation sites.

To determine the specificity of this kinase, we next identified its autophosphorylation sites. A mass spectrometry approach was used since this technique allows precise characterization of posttranslational modifications including phosphorylation (41,[START_REF] Molle | An improved method to unravel phosphoacceptors in Ser/Thr protein kinase-phosphorylated substrates[END_REF]. NanoLC/nanospray/ tandem mass spectrometry (LC-ESI/MS/MS) was applied for the identification of phosphorylated peptides and for localization of phosphorylation sites in CstK. This approach led to 97% of sequence coverage, while the remaining residues uncovered that did not include Ser, Thr or Tyr residues.

As detailed in Table 3, analysis of tryptic digests allowed the characterization of three phosphorylation sites in CstK. Surprisingly, unlike classical Ser/Thr or Tyr kinases, CstK was phosphorylated on two Tyr residues (Y14 and Y209), in addition to one Thr site (T232). Since protein sequence analysis did not reveal a classical activation loop in this kinase, the contribution of T232, Y14 and Y209 to CstK kinase activity was individually assessed. Hence, these residues were mutated either to phenylalanine to replace tyrosine residues or alanine to replace threonine residue, generating the single mutants CstK_Y14F, CstK_Y209F, and CstK_T232A as well as the CstK_Y14F/Y209F/T232A triple mutant (CstK_FFA). Next, in vitro kinase assays with [γ-33 P]ATP were carried out and revealed that maximum loss in CstK autophosphorylation activity was observed in the CstK_Y14F mutant (Fig. 2D), suggesting that this site is central for CstK activation. In contrast, the CstK_Y209F mutant exhibited a slight hyperphosphorylation, which might indicate that Y209 only plays an accessory role in controlling CstK autophosphorylation (Fig. 2D). Finally, the CstK_T232A mutant showed a reduced CstK phosphorylation and displayed diminished kinase activity towards the exogenous substrate MBP (Fig. 2D). Note that mutating all three autophosphorylation sites fully abrogated CstK kinase activity (Fig. 2D). These results indicate that Y14 and T232 are the major phosphorylation sites in CstK and strongly suggest that CstK might be a dual specificity (Thr/Tyr) kinase.

CstK activity and phosphorylated state affect its intracellular localization.

Next, we ectopically expressed HA-tagged CstK, CstK_K55M and CstK_FFA derivatives in noninfected and C. burnetii infected U2OS cells, to investigate its intracellular localization. CstK mainly localized at vesicular compartments in noninfected cells, and accumulated at CCVs upon C. burnetii infection, suggesting an active role in the biogenesis of this compartment (Fig. 1C). Interestingly, the inactive CstK_K55M mutant localized at vesicular structures positive for the lysosomal marker LAMP1 but not at CCVs while the non-phosphorylated CstK_FFA displayed a diffuse localization in the cytosol of transfected cells (Fig. 2E). Overall, these data suggest that the kinase activity and phosphorylated state of CstK play an important role in its localization in cells.

CstK regulates vacuole development and C. burnetii replication within infected cells.

As a first step towards the understanding of CstK functions in the course of infection, and to appreciate the extent to which this kinase is required for growth and viability of C. burnetii, we attempted to inactivate the corresponding chromosomal gene. Unfortunately, after several attempts we were unable to generate a null mutant suggesting that cstK might be essential. However, we had previously isolated a C. burnetii mutant (Tn2496) carrying a transposon insertion allowing GFP expression at position 156783, 32 bp upstream of the starting codon of cstK (43) (Fig. 1A). To determine the effect of this transposon insertion on cstK gene expression, we assessed the expression level of cstK mRNA from wt C. burnetii and Tn2496 strains. Surprisingly, cstK expression was significantly upregulated in the mutant strain, suggesting that the transposon insertion may have released a transcriptional negative regulation (Fig. 3A). This suggested that a putative transcriptional regulator might bind the cstK promoter and control its activity during host invasion.

We next examined the effects of CstK overexpression on C. burnetii infections by challenging Vero cells either with wt C. burnetii, the Dot/Icm-defective dotA::Tn mutant or the Tn2496 mutant. Intracellular growth of the CstK overexpressing strain was significantly reduced over 7 days of infection with an intermediate phenotype between wt and the dotA::Tn mutant (Fig. 3B).

Accordingly, multiparametric phenotypic profile analysis of the Tn2496 mutant indicated that this strain exhibited a major defect in CCV development as compared to wt C. burnetii (Fig. 3C &D). To further investigate the effects of CstK overexpression on C. burnetii infections, GFP-expressing C. burnetii were transformed with plasmids expressing HA-tagged wt CstK or its corresponding mutants (CstK_K55M and CstK_FFA) under the control of an IPTG promoter. U2OS cells expressing cytoplasmic mCherry were challenged with the three C. burnetii strains in the presence or absence of IPTG. After 6 days of infection, cells were fixed, labelled with Hoechst and anti-LAMP1 antibody to visualize host cells nuclei and CCVs respectively, and processed for automated image analysis. In all cases, the overexpression of CstK was detrimental for CCVs biogenesis and bacterial replication (Fig. 3E). Next, U2OS cells were challenged either with wt C. burnetii, the GFP-expressing Tn2496 mutant strain or a combination of the two for 6 days (Fig. 3F). Cells were then fixed and labelled with an anti-C. burnetii antibody to label both bacteria strains and incubated with Hoechst to visualize host cells nuclei (Fig. 3F). Automated image analysis was then used to determine the effects of CstK overexpression on the replication of wt bacteria, in trans. Co-infections resulted in a significant increase in the size of Tn2496 colonies, indicating that wt C. burnetii can partially restore the growth of the CstK-overexpressing strain (Fig. 3G). However, a significant decrease in the size of bacterial colonies labelled by the anti-C. burnetii antibodies indicated that CstK-overexpression has a detrimental effect in trans, on the development of wt bacteria (Fig. 3G). Of note, vacuoles harboring wt or mutant colonies alone were never observed in co-infection experiments. Therefore, we concluded that CstK participates in the formation of the C. burnetii replicative vacuole, and that its expression must be finely tuned for optimal intracellular replication.

CstK specifically interacts with host cell proteins.

Since CstK is a secreted protein, we assume that this kinase would interfere with host cell signal transduction pathways to subvert host cell defenses to the benefit of the bacteria. To identify host cell proteins that could interact with CstK, we made use of the model amoeba Dictyostelium discoideum. D. discoideum is a eukaryotic professional phagocyte amenable to genetic and biochemical studies. Lysate from cells overexpressing CstK tagged with a C-terminal FLAG epitope (CstK-FLAG) was incubated with beads coupled to an anti-FLAG antibody. Beads were extensively washed, and bound proteins were separated by SDS-PAGE before mass-spectrometry analysis. Among the putative interactants of CstK identified by this approach, some were discarded on the basis of their intracellular localization while other retained candidates were mostly involved in the endocytic pathway (Table S1). Among these, the Rab GAP/TBC domain-containing protein, DDB_G0280253 (UniProtKB -Q54VM3), is a 136.4 kDa protein homologous to mammalian TBC1D5 (http://dictybase.org), a GTPaseactivating protein (GAP) for Rab7a and Rab7b (44-46) that acts as a molecular switch between the endosomal and the autophagy pathway [START_REF] Popovic | TBC1D5 and the AP2 complex regulate ATG9 trafficking and initiation of autophagy[END_REF]. Given the recently reported implication of TBC1D5 in the biogenesis of L. pneumophila-containing vacuoles [START_REF] Barlocher | Structural insights into Legionella RidL-Vps29 retromer subunit interaction reveal displacement of the regulator TBC1D5[END_REF] and the role of autophagy in the biogenesis of CCVs [START_REF] Beron | Coxiella burnetii localizes in a Rab7-labeled compartment with autophagic characteristics[END_REF][START_REF] Siadous | Coxiella effector protein CvpF subverts RAB26-dependent autophagy to promote vacuole biogenesis and virulence[END_REF][START_REF] Mcdonough | Host pathways important for Coxiella burnetii infection revealed by genome-wide RNA interference screening[END_REF], we aimed at validating the interaction between human TBC1D5 (Hs-TBC1D5) and CstK in HEK-293T cells. Cells coexpressing Hs-TBC1D5-GFP and HA-CstK or CstK mutants were used for immunoprecipitation using anti-HA beads. Wild-type and CstK derivatives were detected as coimmunoprecipitated in presence of Hs-TBC1D5-GFP, thus confirming that Hs-TBC1D5 is a bona fide CstK interactant (Fig. 4A). Significantly higher levels of TBC1D5 were coimmunoprecipitated by the CstK mutants suggesting that the interaction might be increased in the absence of phosphorylation turnover of the kinase (Fig 4A ,lower panel). Interestingly, the interaction is not dependent on the phosphorylation status of CstK as the K55M mutant or the triple FFA mutant are still able to interact. Other candidates identified by mass spectrometry are currently being investigated.

TBC1D5 is recruited at CCVs and regulates their biogenesis.

Given its recently reported role in the development of L. pneumophila-containing vacuoles, we investigated the localization of TBC1D5 in U2OS cells infected either with wt C. burnetii, the CstK-overexpressing strain Tn2496 or the Dot/Icm-defective mutant dotA::Tn. Consistently with previous studies demonstrating a role in the activation of Rab7a and b, TBC1D5 seems to accumulate specifically at CCVs in a Dot/Icm-independent manner as the eukaryotic protein was found at CCVs generated by all C. burnetii (wt, the Tn2496 and the dotA::Tn Fig. 4B and Supplementary Fig. 1). Next, we used siRNA to deplete cells of TBC1D5 prior to C. burnetii infection, to investigate a possible role in CCVs development and intracellular bacterial replication. Indeed, vacuole development was significantly reduced in cells exposed to Hs-TBC1D5-targeted siRNAs as opposed to cells treated with nontargeting siRNA oligonucleotides (Fig. 4C).

TBC1D5 is not phosphorylated in vitro by recombinant CstK.

We assessed whether CstK might phosphorylate the recombinant Hs-TBC1D5. Despite the in silico prediction of several Ser/Thr and Tyr phosphorylatable residues in Hs-TBC1D5, we failed to detect Hs-TBC1D5 phosphorylation using several in vitro kinases assays (Supplementary Fig. 2). In addition, Hs-TBC1D5 phosphorylation status was also investigated upon transfection with CstK or its inactive derivative (K55M) followed by Hs-TBC1D5 immunoprecipitation. No phosphorylation could be detected in our experimental conditions. Purifications of the GST-tagged recombinants were performed as described by the manufacturer (GE Healthcare). cstK coding sequence was also optimized for mammalian cell expression (GenScript), amplified by PCR and cloned into pDXA-3C ( 50) containing a FLAG-tag for Cterminal fusion. After sequencing, the plasmid was linearized by the restriction enzyme ScaI and transfected in D. discoideum as described [START_REF] Alibaud | Dictyostelium discoideum transformation by oscillating electric field electroporation[END_REF].

DISCUSSION

Clone selection was made with 10 mg/ml of G418, and protein expression was assayed by Western blot analysis of D. discoideum crude extract with an anti-FLAG rabbit polyclonal antibody (GenScript, USA). For ectopic expression assays, cstK, cstK_K55M and cstK_FFA with optimized codons (IDT) were cloned in pRK5-HA using the primer pair CstKopt-BamHI-Fw/CstKopt-EcoRI-Rv.

Cloning, expression and purification of TBC1D5

derivatives. The D. discoideum GFP-tagged TBC1D5 was previously generated [START_REF] Barlocher | Structural insights into Legionella RidL-Vps29 retromer subunit interaction reveal displacement of the regulator TBC1D5[END_REF]. Cells were grown at 22 °C in HL5 medium as previously described [START_REF] Alibaud | Dictyostelium discoideum transformation by oscillating electric field electroporation[END_REF]. Human TBC1D5 coding sequence was obtained from the hORFeome v8.1 (ORF 2659, Q92609, fully-sequenced cloned human ORFs in Gateway Entry clones ready for transfer to Gateway-compatible expression vectors). HsTBC1D5 coding sequence has been recombined into pEGFP-N1 RfC Destination vector by GateWay reaction (MGC Platform Montpellier) thus generating pEGFP-N1_HsTBC1D5 coding for HsTBC1D5 with a C-terminal GFP Tag. pmCH_Hs-TBC1D5-mCherry has been generated by the same method (MGC Platform Montpellier).

RNA extraction and quantitative RT-PCR (qRT-PCR).

Fifty ml of C. burnetii culture was harvested, resuspended in 600 µL of RNA protect reagent (Qiagen) and incubated 5 min at room temperature. Bacteria were centrifuged and resuspended in 200 µl TE (10 mM Tris-HCl, 1 mM EDTA, pH 8) containing 1 mg/ml lysozyme. Bacterial suspension was incubated at room temperature for 5 min and bacteria were disrupted by vigorous vortexing for 10 sec every 2 min. 700 µl of RLT buffer from RNA easy kit (Qiagen), were added to the bacterial suspension and disruption was completed by vortexing vigorously. RNA was purified with the RNA easy kit according to manufacturer's instructions. DNA was further removed using DNAse I (Invitrogen). cDNA was produced using Superscript III reverse transcriptase (Invitrogen). Controls without reverse transcriptase were done on each RNA sample to rule out possible DNA contamination. Quantitative real-time PCR was performed using LightCycler 480 SYBR Green I Master (Roche) and a 480 light cycler instrument (Roche). PCR conditions were as follows: 3 min denaturation at 98 °C, 45 cycles of 98 °C for 5 sec, 60 °C for 10 sec and 72 °C for 10 sec. The dotA gene was used as internal control. The sequences of primers used for qRT-PCR are listed in Table 2. The expression level of cstK in the wild type strain was set at 100 and the expression levels of cstK in the Tn2496 mutant were normalized to the wild type levels.

In vitro kinase assays. In

Mass spectrometry analysis.

For mass spectrometry analysis, CstK was phosphorylated as described above, except that [γ-33 P]ATP was replaced with 5 mM cold ATP. Subsequent mass spectrometry analyses were performed as previously reported [START_REF] Brelle | Phosphorylation-mediated regulation of the Staphylococcus aureus secreted tyrosine phosphatase PtpA[END_REF][START_REF] Baronian | Phosphorylation of Mycobacterium tuberculosis ParB participates in regulating the ParABS chromosome segregation system[END_REF]. Briefly, samples were submitted to trypsin digestion and analyzed using an Ultimate 3000 nano-RSLC (Thermo Scientific, San Jose California) coupled on line with a quadrupole-orbitrap Q Exactive HF mass spectrometer via a nano-electrospray ionization source (Thermo Scientific, San Jose California).

Samples were injected and loaded on a C18 Acclaim PepMap100 trap-column (Thermo Scientific) and separated on a C18 Acclaim Pepmap100 nano-column (Thermo Scientific). MS data were acquired in a data dependent strategy selecting the fragmentation events based on the 20 most abundant precursor ions in the survey scan (350-1600 Th). The resolution of the survey scan was 60,000 at m/z 200 Th and for MS/MS scan the resolution was set to 15,000 at m/z 200 Th.

Peptides selected for MS/MS acquisition were then placed on an exclusion list for 30s using the dynamic exclusion mode to limit duplicate spectra. Data files were then analyzed with Proteome Discover 2.2 using the SEQUEST HT algorithm against the Uniprot Dictyostelium discoideum to which was included the sequence of CstK.

C. burnetii infections. U2OS epithelial cells were challenged either with Coxiella burnetii RSA439 NMII, the transposon mutants Tn1832, dotA::Tn or Tn2496 as previously described [START_REF] Hanks | The protein kinase family: conserved features and deduced phylogeny of the catalytic domains[END_REF][START_REF] Mcdonough | Host pathways important for Coxiella burnetii infection revealed by genome-wide RNA interference screening[END_REF] [START_REF] Martinez | Generation and multi-phenotypic high-content screening of Coxiella burnetii transposon mutants[END_REF]. Samples were imaged with a Zeiss Axio Imager Z1 epifluorescence microscope (Carl Zeiss, Germany) connected to a CoolSNAP HQ 2 CCD camera (Teledyne Photometrics, Tucson, AZ). Images were acquired with 40x oil immersion objectives and processed with Metamorph (Molecular Devices, San Jose, CA). For phenotypic screening, samples were imaged with an ArrayScan VTI Live epifluorescence automated microscope (Cellomics) equipped with an ORCA-ER CCD camera (Hamamatsu). 25 fields per well were acquired for image analysis. ImageJ and ICY software were used for image analysis and quantifications. Phenotypic profiles (expressed as z-scores) were calculated using CellProfiler, from triplicate experiments as previously described [START_REF] Martinez | Generation and multi-phenotypic high-content screening of Coxiella burnetii transposon mutants[END_REF] following median based normalization of 96-well plates. Plates effects were corrected by the median value across wells that are annotated as control.

Beta-lactamase translocation assay.

Effector proteins translocation assays were performed as previously described [START_REF] Martinez | Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development[END_REF]. Briefly, Coxiella burnetii Tn1832 (wt) and dotA::Tn were transformed either with pXDC-Blam (negative control), pXDC-Blam-CvpB (positive control) or pXDC-Blam-CstK. Each strain was used to infect U2OS epithelial cells. After 24, 48 or 72 hours of infection, cells were loaded with the fluorescent substrate CCF4/AM (LiveBLAzer-FRET B/G loading kit; Invitrogen) in HBSS (20 mM HEPES pH 7.3) containing 15 mM probenecid (Sigma). Cells were incubated in the dark for 1 h at room temperature and imaged using an EVOS inverted fluorescence microscope. Images were acquired using DAPI and GFP filter cubes. The image analysis software CellProfiler was used to segment and identify all cells in the sample (GFP), positive cells (DAPI) and to calculate the intensity of fluorescence in each channel. The percentage of positive cells versus the total number of infected cells was then calculated and used to evaluate effector translocation.

Immunoprecipitation from D. discoideum lysates.

For immunoprecipitation assays, 2x10 7 cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.5% NP40, protease inhibitors (Roche)) and cleared by centrifugation for 15 min at 14 000 rpm in a microfuge. Lysate supernatants were incubated overnight at 4 °C with anti-flag monoclonal antibody coated on agarose beads (Genscript). The beads were then washed five times in wash buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.1% NP40) and once in PBS. Bound proteins were migrated on SDS-PAGE and analyzed by LC-MS/MS.

Cell culture, heterologous expression and anti-HA immunoprecipitation.

HEK-293T cells were grown in DMEM (Gibco) containing 10% (vol/vol) FBS, 1% glutamax (Gibco, 200 mM stock), 0.5% Penicillin/Streptomycin (Gibco, 10000 U/mL stock) and maintained under standard conditions at 37 °C in a humidified atmosphere containing 5% CO2. Cells were transiently transfected using the jetPEI Transfection Reagent (Polyplus-Transfection Inc.) to express either Hs-TBC1D5-GFP, CstK_HA derivatives, or each CstK derivatives with TBC1D5_GFP protein. Cells were used 24h after transfection for immunoprecipitation assay. Transfected cells were washed two times in cold PBS and lysed in lysis buffer (50 mM Tris, 150 mM NaCl, 0.5 mM EDTA, 0.5 % NP40, protease and phosphatases inhibitors (Roche)). Cleared lysate (950 µL, approximately 1 mg total proteins) were incubated with anti HA magnetic Beads (Pierce) for 30 min at room temperature under gentle rotation. The beads were washed three times in lysis buffer, boiled in 2X Laemmli sample buffer and loaded on ExpressPlus™ PAGE Gels (GenScript). The eluted proteins were visualized by Western Blotting with the following antibodies: anti-HA from Chromotek, anti-GFP from Torrey Pines, donkey anti-rat or anti-rabbit from Jackson ImmunoResearch. Values are means ± SEM of 4 independent experiments. ***, p < 0.001, unpaired t-test. (B) Vero cells were challenged either with wt C. burnetii, the dotA::Tn mutant or the Tn2496 mutant strains, all expressing GFP. Intracellular replication was monitored over 7 days of infection by monitoring the GFP fluorescence using a microplate reader. Values are mean ± SD from 3 independent experiments. (C) U2OS cells were challenged either with wt C. burnetii or the Tn2496 mutant strain, both expressing GFP (green). 7 days post-infection, cells were fixed and labelled with an anti-LAMP1 antibody (red) and Hoechst (blue) to visualize CCVs and host cell nuclei, respectively. Scale bars 10 μm. (D) An average of 50000 cells were automatically imaged and analyzed from triplicate experiments for each condition illustrated in C, and the phenotypic profile of the Tn2496 mutant was compared to that of wt C. burnetii and expressed as z-scores over 15 independent features. (E) GFP-expressing C. burnetii transformed with plasmids encoding either wt CstK, the K55M or the FFA mutants, all under the regulation of an IPTG-inducible promoters were used to infect U2OS cells for 6 days, in the presence or absence of IPTG. Cells were then fixed and labelled as in C, and an average of 50000 cells were automatically imaged and analyzed for each condition. The mean size of C. burnetii colonies and CCVs was calculated (red bars). (F) U2OS cells were challenged either with wt C. burnetii, the GFP-expressing mutant Tn2496 or a combination of the two strains for 6 days. Cells were then fixed and labelled with anti-C. burnetii antibodies (anti-NMII) to label both strains. (G) An average of 50000 cells were automatically imaged and analyzed for each condition illustrated in F and the mean size of C. burnetii colonies was calculated (red bars). n.s., non-significant; ****, p < 0.0001, one-way ANOVA, Dunnett's multiple comparison test.

TABLES

Figure 1 .

 1 Figure 1. CstK is a Dot/Icm effector protein (A) Schematic representation of CstK. A conserved promoterbinding domain is predicted between aa -227 and -213 (Apm) and the transposon insertion site for Tn2496 is indicated. The kinase domain is predicted between aa 26 and 174 and is shown in red. The conserved lysine residue is indicated, and the phosphorylated sites are indicated by an asterisk. (B) BLAM secretion assay. U2OS cells were infected for 24, 48, or 72h (black, grey and white bars, respectively) with wt C. burnetii or the dotA::Tn mutant transformed with vectors expressing Beta-Lactamase alone (BLAM, negative control), BLAM-CvpB (positive control) or BLAM-CstK. Protein translocation was probed using the CCF-4 substrate. The average percentage of cells positive for cleaved CCF-4 as compared to the total number of cells was assessed. Values are mean ± SD from 3 independent experiments. ns = non-significant; *** = P<0.0001, one-way ANOVA, Dunnett's multiple comparison test. CstK localization. (C) U2OS cells ectopically expressing HA-tagged CstK (red) were either left untreated (top panels) or challenged with wt C. burnetii expressing GFP (green) for 3 days. Lysosomal compartments were labelled using anti-LAMP1 antibodies (blue). Scale bars 10 μm.

Figure 2 .

 2 Figure 2. (A) Biochemical characterization of CstK. Recombinant CstK derivatives were overproduced, purified on glutathione-Sepharose 4B matrix and submitted to gel electrophoresis and stained with Coomassie Blue (upper panel). In vitro phosphorylation assays were performed with [γ-33 P]ATP for 30 min and the eukaryotic substrate myelin basic protein (MBP) when required. Proteins were analyzed by SDS-PAGE, and radioactive bands were revealed by autoradiography (lower panel). The upper bands illustrate the autokinase activity of CstK, and the lower bands represent phosphorylated MBP. Standard proteins of known molecular masses were run in parallel. (B) A kinetic analysis was performed by incubation of CstK with [γ-33 P]ATP over different times. Proteins were analyzed by SDS-PAGE, and radioactive bands were revealed by autoradiography. (C) Effects of cations on autokinase activity of CstK. Purified CstK protein was subjected to in vitro autophosphorylation assays in the presence of 50 μM ATP [γ-33 P]ATP and 5 mM of Mg 2+ or Mn 2+ . Phosphoproteins were separated by SDS-PAGE and then revealed by autoradiography. (D) In vitro phosphorylation of CstK mutant derivatives. The mutated variants CstK_Y14F (harboring a Tyr to Phe substitution at Y14), CstK_Y209F (harboring a Tyr to Phe substitution at Y209), CstK_T232A (harboring a Thr to Ala substitution at T232), or CstK_Y14F/Y209F/T232A (harboring all three substitutions) were incubated in presence of [γ-33 P]ATP with or without the eukaryotic substrate myelin basic protein (MBP). Samples were separated by SDS-PAGE, stained with Coomassie Blue (upper panel), and visualized by autoradiography (lower panel). The upper bands illustrate the autokinase activity of CstK, and the lower bands represent phosphorylated MBP. Standard proteins of known molecular masses were run in parallel (kDa lane). (E) U2OS cells ectopically expressing either HA-tagged CstK_K55M or CstK_FFA (red) were challenged with wt C. burnetii expressing GFP (green) for 3 days. Lysosomal compartments were labelled using anti-LAMP1 antibodies (blue). Scale bars 10 μm. Inset magnification scale bars 4 μm.

Figure 3 .

 3 Figure 3. CstK regulates vacuole development and C. burnetii replication within infected cells. (A)Expression of cstk was measured by qRT-PCR from cultures of either wt C. burnetii or the Tn2496 mutant strain. Values are means ± SEM of 4 independent experiments. ***, p < 0.001, unpaired t-test. (B) Vero cells were challenged either with wt C. burnetii, the dotA::Tn mutant or the Tn2496 mutant strains, all expressing GFP. Intracellular replication was monitored over 7 days of infection by monitoring the GFP fluorescence using a microplate reader. Values are mean ± SD from 3 independent experiments. (C) U2OS cells were challenged either with wt C. burnetii or the Tn2496 mutant strain, both expressing GFP (green). 7 days post-infection, cells were fixed and labelled with an anti-LAMP1 antibody (red) and Hoechst (blue) to visualize CCVs and host cell nuclei, respectively. Scale bars 10 μm. (D) An average of 50000 cells were automatically imaged and analyzed from triplicate experiments for each condition illustrated in C, and the phenotypic profile of the Tn2496 mutant was compared to that of wt C. burnetii and expressed as z-scores over 15 independent features. (E) GFP-expressing C. burnetii transformed with plasmids encoding either wt CstK, the K55M or the FFA mutants, all under the regulation of an IPTG-inducible promoters were used to infect U2OS cells for 6 days, in the presence or absence of IPTG. Cells were then fixed and labelled as in C, and an average of 50000 cells were automatically imaged and analyzed for each condition. The mean size of C. burnetii colonies and CCVs was calculated (red bars). (F) U2OS cells were challenged either with wt C. burnetii, the GFP-expressing mutant Tn2496 or a combination of the two strains for 6 days. Cells were then fixed and labelled with anti-C. burnetii antibodies (anti-NMII) to label both strains. (G) An average of 50000 cells were automatically imaged and analyzed for each condition illustrated in F and the mean size of C. burnetii colonies was calculated (red bars). n.s., non-significant; ****, p < 0.0001, one-way ANOVA, Dunnett's multiple comparison test.

Figure 4 .

 4 Figure 4. CstK interacts with TBC1D5. (A) The interaction between CstK and the human TBC1D5 has been confirmed after cotransfection of HEK-293T cells to express each HA-tagged CstK derivatives with GFP-tagged Hs-TBC1D5. Cells have been lysed and HA-tagged CstK derivatives have been trapped on anti-HA magnetic beads (Pierce). Beads were washed, eluted by boiling, and bound proteins were revealed by Western blot analysis. Anti-HA antibody confirms the immunoprecipitation of CstK derivatives. For the densitometry graph, Regions of Interest (ROIs) were obtained from each band of interest and the intensity was measured (B) Hs_TBC1D5 localization during infection was monitored in U2OS cells expressing mCherry-tagged Hs-TBC1D5 (red) challenged for 4 days either with wt C. burnetii, the Dot/Icm-defective dotA::Tn mutant or the CstK-overexpressing mutant Tn2496, all expressing GFP (green). Lysosomal compartments were labelled with anti-LAMP1 antibodies (blue). Scale bars 10 μm. Inset magnification scale bars 4 μm (C) The role of TBC1D5 in C. burnetii infections was investigated using siRNA to deplete Hs-TBC1D5 in U2OS prior to challenge with GFP-expressing wt C. burnetii. The size of CCVs was automatically calculated over an average of 150000 cells per condition. Red bars indicate medians. ***, p < 0.0001; **, p < 0.001; *, p < 0.01; one-way ANOVA, Dunnett's multiple comparison test. Scale bars 10 μm.

Table 3 .

 3 Figure 1

Cloning, expression and purification of CstK derivatives.

  Bacterial Ser/Thr/Tyr kinases expressed by pathogenic bacteria can either act as key regulators of important microbial processes, or be translocated by secretion systems to interact with host substrates, thereby our results provide the first biochemical analysis of the secreted C. burnetii kinase CstK and its involvement in the process of infection and CCVs development. Importantly, CstK presents important differences as compared to classical Ser/Thr kinases. In particular, we provided evidence that CstK is a dual kinase able to autophosphorylate on Thr and Tyr residues. Moreover, the observation that a transposon insertion 32 bp upstream of the cstK starting codon leads to an increase in the levels of cstK mRNAs was indicative of the presence of a negative transcriptional regulation of gene expression, suggesting a fine tuning of the levels of CstK. Indeed, the Tn2496 mutant displays a severe CCV biogenesis defect when used to challenge U2OS cells, highlighting the importance of regulating cstK expression during C. burnetii infections. The cstK (CBU_0175) gene was amplified by PCR using Coxiella burnetii RSA439 NMII chromosomal DNA as a template with the primers listed in Table2containing a BamHI and

	Transformed E. coli BL21 Star cells with	Indeed TBC1D5 associates the retromer VPS29
	pGEX(M)_cstK derivatives were grown at 16 °C in	subunit involved in endosomal trafficking, and
	LB medium containing 1 g/liter of glucose and 100	upon autophagy induction, the autophagy
	µg/ml of ampicillin and protein synthesis induced	ubiquitin-like protein LC3 can displace VPS29,
	with 0.5 mM IPTG overnight. Bacteria were	thus orienting TBC1D5 functions towards
	disrupted by sonication (Branson, digital sonifier)	autophagy instead of endosomal functions (47). It
	and centrifuged at 14,000 rpm for 25 min.	is thus tempting to propose that CstK might
		interfere with this tight regulation between
		TBC1D5, LC3 and VPS29, and redirect TBC1D5
		functions to support efficient C. burnetii
		intracellular replication. Further work will need to
		be carried out to decipher how CstK recognizes
		these host substrates and how they participate in
		the establishment of C. burnetii parasitophorous
		vacuoles. Another perspective of this work is the
		opening of a new field of investigation for future
		drug development to fight this pathogen. Because
		CstK seems to be essential, specific inhibitors of
		this kinase preventing C. burnetii growth would be
		extremely useful for the development of new
		therapies.
		EXPERIMENTAL PROCEDURES
		Bacterial strains and growth conditions. Bacterial
		strains and plasmids are described in Table 1.
		Strains used for cloning and expression of
	Accordingly, inducing the expression of wt CstK	recombinant proteins were Escherichia coli
	in wt C. burnetii severely impairs CCVs	TOP10 (Invitrogen) and E. coli BL21 (DE3)Star
	development and bacterial replication. Co-	(Stratagene), respectively. E. coli cells were grown
	infection experiments demonstrated that CstK	and maintained at 25 °C in LB medium
	overexpression can also act in trans, by perturbing	supplemented with 100 µg/ml ampicillin when
	the intracellular replication of wt C. burnetii. The	required. Coxiella burnetii RSA439 NMII and
	identification of candidate eukaryotic interactors of	transposon mutants Tn1832, Tn2496, and dotA::Tn
	CstK further corroborated a role of the bacterial	were grown in ACCM-2 (45) supplemented with
	kinase in subverting host functions during	chloramphenicol (3 mg/ml) in a humidified
	infection. Here we confirmed that CstK interacts	atmosphere of 5% CO2 and 2.5% O2 at 37 °C.
	with TBC1D5, but we failed to detect
	phosphorylation of the eukaryotic target by CstK.
	Though we cannot exclude that TBC1D5 is a
	genuine CstK substrate in vivo as the lack of
	phosphorylation of host interactors of bacterial
	STPKs is not uncommon. Interaction between
	STPKs and host proteins might as well perturb	HindIII restriction site, respectively. The
	protein interaction networks at play in host cells	corresponding amplified product was digested with
	(21). Indeed, the induced overexpression of CstK	BamHI and HindIII, and ligated into the bacterial
	mutants lacking kinase activity in wt C. burnetii	pGEX(M) plasmid, that includes a N-terminal
	impaired CCVs development to the same extent as	GST-tag, thus generating pGEX(M)_cstK.
	the overexpression of wt CstK. The biochemical	pGEX(M)_cstK derivatives harboring different
	mechanisms of these pathogen-directed targeted	mutations were generated by using the
	perturbations of host cell-signaling networks are	QuikChange Site-Directed Mutagenesis Kit
	being actively investigated. Regardless, siRNA
	depletion of TBC1D5 in C. burnetii-infected cells
	points at a role of the eukaryotic protein in CCVs
	development. In mammals, TBC1D5 was
	suggested to function as a molecular switch
	between endosomal and autophagy pathways.

(Stratagene, La Jolla, CA), and resulted in the construction of plasmids detailed in Table

2

. For overexpression assays, cstK and its derivatives were cloned in the pJA-LACO-4xHA vector

[START_REF] Martinez | Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development[END_REF]

using KpnI and BamHI restriction sites. All constructs were verified by DNA sequencing.

Table 1 . Strains and plasmids used in this study Strains or Plasmids Genotype or Description a Source or Reference

 1

	Escherichia coli strains
	E. coli TOP10	E. coli derivative ultra-competent cells	Invitrogen
		used to general cloning; F-mcrA
		D(mrr-hsdRMS-mcrBC)
		f80dlacZÄM15 D lacX74 endA1
		recA1araD139 D (ara, leu)7697 galU
		galK rpsL nupG -tonA
	E. coli BL21(DE3)Star	F2 ompT hsdSB(rB2 mB2) gal dcm	Stratagene
		(DE3); used to express recombinant
		proteins in E. coli
	Coxiella burnetii strains
	Coxiella burnetii RSA439 NMII	Wild type (wt) Coxiella burnetii	(55)
		RSA439 NMII non-fluorescent
	Coxiella burnetii Tn2496	Coxiella burnetii RSA439 NMII	This study
		carrying	an	Himar1-CAT-GFP
		cassette 32 bp upstream of CBU_0175,
		expressing GFP
	Coxiella burnetii Tn1832	Coxiella burnetii RSA439 NMII	(43)
		carrying	an	Himar1-CAT-GFP
		cassette in the intergenic region
		between CBU_1847b and CBU_1849,
		expressing GFP
	Coxiella burnetii Tn292	Coxiella burnetii RSA439 NMII	(43)
		carrying	an	Himar1-CAT-GFP
		cassette in CBU_1648 (dotA),
		expressing GFP
	Plasmids
	pGEX(M)	pGEX with a 321-bp EcoRI/BamHI	(56)
		fragment from pET19b introducing a
		HindIII site in the pGEX polylinker
	pGEX(M)_cstK	pGEX(M) derivative used to express	This study
		GST-tagged fusion of CstK (Amp R )
	pGEX(M)_cstK_K55M	pGEX(M) derivative used to express	This study
		GST-tagged fusion of CstK_K55M
		(Amp R )
	pGEX(M)_cstK_Y14F	pGEX(M) derivative used to express	This study
		GST-tagged fusion of CstK_Y14F
		(Amp R )
	pGEX(M)_cstK_Y209F	pGEX(M) derivative used to express	This study
		GST-tagged fusion of CstK_Y209F
		(Amp R )

Table 2 . Primers used in this study Primers 5' to 3' Sequence a, b

 2

	NtermCstK	TATGGATCCTTAATGGCTTATATGAGGCTTAGT (BamHI)
	CtermCstK	TATAAGCTTTTAATCCCATTCAATATTTTC (HindIII)
	RvCstK K55M	ATAAAAGAGCATCGCCGCTCG

ACKNOWLEDGMENTS

We wish to thank the Montpellier RIO imaging facility at the University of Montpellier, member of the national infrastructure France-BioImaging, that is supported by the French National Research Agency (ANR-10-INBS-04, «Investments for the future»). We acknowledge the contribution of the Protein Science Facility of the SFR Biosciences (UMS3444/CNRS, US8/Inserm, ENS de Lyon, UCBL), especially Frédéric DELOLME and Adeline PAGE who performed the mass spectrometry analysis. This work was supported by grants from the ATIP/AVENIR Program for V.M. and M.B., the Region Occitanie for S. B., a Marie Curie CIG to E.M. (grant n. 293731), the Agence Nationale de la Recherche (ANR) Grant ANR-14-CE14-0012-01 (project AttaQ) to M.B.

DATA AVAILABILITY STATEMENT

All the data are contained in this manuscript.

Densitometry.

Regions of Interest (ROIs) were obtained from each band of interest and the intensity was measured using ImageLab (From Biorad). For each band, the same ROI was used for background calculation and removal from areas adjacent to each band.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest with the contents of this article.


A Kisrane-Bouzidi

Mosbah Zidani

M C C Nebbar

T Abid

A L L Helbert

Francois Brisset

Thierry Baudin

Mechanical Properties and Texture Evolution of High-Carbon Steel Wires during Wire Drawing: Strand Manufacturing

Keywords: High-carbon steel, Wire drawing, Strand, Texture, Mechanical properties

published or not. The documents may come

Introduction

Cold drawing pearlitic steel wires of eutectoid or hyper-eutectoid compositions have been studied extensively as a topic of considerable amounts of scientific researches over the past several decades because of their unique combination of very high strength, torsional and bending strength, reasonable ductility and acceptable level of toughness aside from the other inherent specific material properties such as corrosion resistance, weldability, and machinability [START_REF] Elwazri | The effect of microstructural characteristics of pearlite on the mechanical properties of hypereutectoid steel[END_REF][START_REF] Guo | Microstructure and texture evolution in fully pearlitic steel during wire drawing[END_REF][START_REF] Borchers | Cold-drawn pearlitic steel wires[END_REF][START_REF] Languillaume | Cementite Dissolution in Heavily Cold Drawn[END_REF][START_REF] Modi | Effect of interlamellar spacing on the mechanical properties of 065% C steel[END_REF][START_REF] Xiang | Chloride-induced corrosion behavior of cold-drawn pearlitic steel wires[END_REF]. They are largely used for many reliability-critical applications including strands, suspension bridge cables, civil engineering and construction, automotive tyre cord, engineering springs, railroad rails, and many other industries [START_REF] Guo | Microstructure and texture evolution in fully pearlitic steel during wire drawing[END_REF][START_REF] Zhou | Effects of Chromium Additions upon Microstructure and Mechanical Properties of Cold Drawn Pearlitic Steel Wires[END_REF][START_REF] Fang | Microstructure and mechanical properties of cold-drawn pearlitic wires affect by inherited texture[END_REF][START_REF] Sahay | Pearlitic Steels: Wire Drawing[END_REF][START_REF] Schmitt | Micromechanical modelling of the elastoplastic behavior of polycrystals containing precipitates -Application to hypo-and hyper-eutectoid steels[END_REF][START_REF] Goto | Application of cold drawn lamellar microstructure for developing ultra-high strength wires[END_REF].

The drawn wires are typically produced by wire drawing technique, which is one of the most frequently applied techniques in the wire manufacturing industry. This process is one of the oldest metal forming processes [START_REF] Fang | Microstructure and mechanical properties of cold-drawn pearlitic wires affect by inherited texture[END_REF][START_REF] Zidani | Deformation textures in wire drawn perlitic steel[END_REF], During the wire drawing operation, the steel wires are cold drawn for cross-sectional reduction by passing through multiple stages of dies [START_REF] Sahay | Pearlitic Steels: Wire Drawing[END_REF][START_REF] Singh | Development of a virtual wire drawing tool for process analysis and optimization[END_REF][START_REF] Zidani | Study of the relation between microstructure and properties (mechanical/electrical) of copper wire drawing and annealed[END_REF]. wire drawing improves the mechanical properties of the wires. This has been attributed on the one hand, to the development of dislocation density, the re-orientation of cementite lamellae into the drawing direction. On the other hand, the reduction of interlamellar spacing and the refinement of grains size.

The strength of the steel wires also increased during wire drawing. Embry and fisher. [START_REF] Embrury | The structure and properties of drawn pearlite[END_REF] proposed that strengthening of wires followed the Hall-Petch relationship which suggests that the strength can be increased by reducing lamellar spacing and by increasing strain [START_REF] Yang | Nanostructure and torsional ductility of heavily cold drawn steel wires[END_REF]. Recently, it has been found that the tensile strength of heavily cold drawn pearlitic wires can be reached by 7 GPa at higher deformation [START_REF] Borchers | Cold-drawn pearlitic steel wires[END_REF][START_REF] Wei | Microstructure and mechanical properties of heavily cold drawn pearlitic steel wires: Effects of low temperature annealing[END_REF][START_REF] Guelton | Microstructure -Property Relationship in Cold-Drawn Pearlitic Steel Wires[END_REF]. Also, numerous investigations [START_REF] Guo | Microstructure and texture evolution in fully pearlitic steel during wire drawing[END_REF][START_REF] Fang | Microstructure and mechanical properties of cold-drawn pearlitic wires affect by inherited texture[END_REF][START_REF] Zidani | Deformation textures in wire drawn perlitic steel[END_REF][START_REF] Embrury | The structure and properties of drawn pearlite[END_REF][START_REF] Kumar | Progressive Changes in the Microstructure and Texture in Pearlitic Steel during Wire Drawing[END_REF][START_REF] Nebbar | Microstructural Evolutions and Mechanical Properties of Drawn Medium Carbon Steel Wire[END_REF][START_REF] Zelin | Microstructure evolution in pearlitic steels during wire drawing[END_REF] have reported that the cold drawing pearlitic wires acquires a textured fibrous microstructure, especially the majority fiber texture <110> in the ferrite phase. As drawing proceeds, this fiber developed and become intensified at higher deformation [START_REF] Embrury | The structure and properties of drawn pearlite[END_REF].

In this work, we investigated the mechanical behavior and texture evolution of cold-drawn pearlitic steel wires produced for strand manufacturing, which were drawn from medium strain to high strain. Also, the correlation between microstructure and mechanical properties of the pearlitic steel wires during wire drawing is discussed.

Experiments

Material and Methods

The present work was carried out on hyper-eutectoid steel wire intended for the manufacturing of strands received by Trefisoud company (The National Company of Wire Drawing and Manufacture of Welding Products, Algeria). This material was commercially produced fully pearlitic steel in the as-rolled condition and was selected as the reference material (wire rod) in the present investigation. The detailed chemical composition of the steel (in weight %) is given in Table1.

The initial perform for wire drawing was in the form of a circular rod of diameter (11.00 mm). This rod was cold drawn to the final wire by eight paths through several dies at room temperature under the dry condition with an average area reduction of 20% in each pass machine. This process resulted in wire with a final diameter of (4.03 mm). During each stage the true strain is calculating by equation (1) :

ε = 2 ln(𝑑 0 /d) (1)
Where 𝐝 𝟎 is the initial diameter, and d the diameter of the wire after successive drawing.

For this investigation, a various of drawn wires specimens were chosen, but results of only 2 strain levels are presented for the current study, (i.e. ε =1.16 medium strain and ε =2.01 high strain). The parameters characterizing the deformation of specimens are given in Table2.

CMn

Table2. Information about specimens investigated in this study, wire diameter 𝐝 𝐢 , strain level ε (Di), drawing strain (.

CMn

The Microstructure features of specimens were examined along the longitudinal wire section after conventional mechanical polishing with SiC papers from 120# to 4000# grit and final thinning was carried out by the use of a tissue paper with the addition of diamond paste. This mechanical polishing followed by etching using a 4% Picral solution (4g picric acid and 96% ethanol). The microstructures were observed through a (VEGA3 TESCAN) scanning electron microscope (SEM) coupled with modern electron optics based on a unique four-lens wide field optics™ design with a proprietary intermediate lens. The accelerating voltage was 20 kV. Microhardness measurements were performed on the longitudinal wire section using Vickers microhardness tester with an applied load of (500g). Tensile tests of specimens were performed at room temperature using a universal material testing machine at Trefisoud company. Tensile strength (TS), yield strength (0.2% proof stress) and elongation were directly determined from their true stress-strain curves. The same specimens were analyzed by SEM-EBSD on a Zeiss 940 SEM with a tungsten filament equipped with an OIM™ camera (Orientation Imaging Microscopy) software. This analysis was performed in the standard manner.

Results and Discussion

Microstructure Evolution

Figures 1(a-b) show the micrographs obtained from the longitudinal section of the wires at different levels of wire drawing. The microstructure of the initial wire (wire rod) is composed of fully pearlitic colonies with different sizes of grains (fig. 1a). Also, the cementite lamellae in pearlitic colonies are randomly oriented and do not have a strong tendency, on the contrary it takes various ways. Zidani et al. [START_REF] Zidani | Deformation textures in wire drawn perlitic steel[END_REF] have noted that this random orientation of the cementite lamellae leads to a heterogeneous microstructure in the section of wires [START_REF] Zidani | Deformation textures in wire drawn perlitic steel[END_REF][START_REF] Kumar | Progressive Changes in the Microstructure and Texture in Pearlitic Steel during Wire Drawing[END_REF][START_REF] Nebbar | Microstructural Evolutions and Mechanical Properties of Drawn Medium Carbon Steel Wire[END_REF].

During cold drawing, the pearlite colonies without a clear preference stretched and aligned along the drawing axis, this elongation is more clear when the strain level increases (fig. 1b), Zhang et al. [START_REF] Zhang | Microstructure and strengthening mechanisms in cold-drawn pearlitic steel wire[END_REF] and He et al. [START_REF] He | Effect of microstructure evolution on anisotropic fracture behaviors of cold drawing pearlitic steels[END_REF] have observed that during wire drawing the cementite lamellae are rotated toward the drawing axis and the thickness of lamellae further decreases when strain level increases, this phenomenon leads to somewhat fibrous structure [START_REF] Fang | Microstructure and mechanical properties of cold-drawn pearlitic wires affect by inherited texture[END_REF][START_REF] Kumar | Progressive Changes in the Microstructure and Texture in Pearlitic Steel during Wire Drawing[END_REF][START_REF] Yang | Nanostructure and mechanical properties of heavily colddrawn steel wires[END_REF]. Moreover, the wire drawing had a strong influence on the interlamellar spacing. As drawing strain increased the interlamellar spacing of cementite decreased and this is clear in the wire deformed to the higher strain level (ε = 2.01). Embury and Fisher. [START_REF] Embrury | The structure and properties of drawn pearlite[END_REF] have already confirmed this study with a simple relationship between lamellae width and true strain [START_REF] Borchers | Cold-drawn pearlitic steel wires[END_REF]:

𝑟 ε = 𝑟 0 exp(-ε 2 ⁄ ) (2)
This relationship has a simple read which was the interlamellar spacing is reduced proportionally to the wire diameter during the wire drawing process.

The grains size evolution of the drawing wires is illustrated in Fig. 2b-c, the microstructure is taken by the EBSD technique. The EBSD data were then treated with the OIM Analysis software, developed by the EDAX company. The cleanup procedure used to analyze the data was a Grain Dilation (one iteration, minimum grain size = 5, grain tolerance angle = 5) followed by a Grain CI Standardisation (same parameters). Also, only the points with a confidence index higher than 0.05 were taken into account [START_REF] Hary | Investigation of the relationships between mechanical properties and microstructure in a Fe-9 % Cr ODS steel[END_REF]. Figure 2b shows the grain color map of the wire rod (ε = 0.00), in this Map all of the grains have a random distribution and the grains have different size with different diameter, on the other hand, Figure 2c shows the grain color map of the deformed wire (ε = 2.01). It can be seen clearly that after wire drawing there is a redistribution of grains along the wire drawing direction and the size of the grains is refined and became smaller.

Figure 2a presents the curve of grain size (diameter) in area fraction. The results of the curve reinforce the previous observation (Fig. 2b-c). Whereas, it can be seen that for the starting material 10 to 11% of area fraction of grains has a diameter varying between 16.19 and 20.06m. While 11 to 13% of area fraction of grains has only a diameter varying between 3.27 and 5.08m for the final drawing wire. Finally, we can say that the increase of the wire drawing is accompanied certainly with the reduction of the grain size.

Mechanical Properties

To investigate the influence of the wire drawing process on the mechanical properties of the pearlitic steel wires we established a tensile test at room temperature for the specimens at a different stage of wire drawing (fig. 3). Thus, a microhardness measurement of the specimens was performed. The detailed results are listed in table 3.

Pearlite colonies

Figure 3 shows the tensile test curve of the wire rod as well as the drawing wires. It is observed that the tensile strength increases with the increase of strain level. The tensile strength of the wire rod ( ε = 0.00) was measured and found it be around 1242 MPa. This tensile strength is increased largely and reached 2618 MPa at higher strain (ε = 2.01). Indeed, the ductility is reduced with the increase of the wire drawing. It can be seen that there is almost no plastic deformation at the final wire. It is well known that work hardening rate is dependent on tensile strength and deformation, and this leads certainly to the rising of work hardening rate as a function of drawing strain [START_REF] Yang | Nanostructure and torsional ductility of heavily cold drawn steel wires[END_REF][START_REF] Djimaoui | Study of microstructural and mechanical behavior of mild steel wires cold drawn at TREFISOUD[END_REF][START_REF] Yamasaki | The microstructure and mechanical properties of drawn and aged pearlitic steel wires[END_REF].

Microhardness, tensile strength and 0.2% yield strength of the steel specimens are plotted as a function of the strain levels in fig. 4. As pointed out by Goto et al. [START_REF] Goto | Application of cold drawn lamellar microstructure for developing ultra-high strength wires[END_REF] and Langford. [START_REF] Langford | Deformation of pearlite[END_REF] in their studies the values of mechanical properties rise exponentially with the increase of strain level. However, as drawing wire increases the elongation decreases and recorded very low values especially in the final wire as shown in fig. 5. Furthermore, the curve of microhardness as a function of strain level (fig. 4) shows that the wire drawing affects the microhardness. It can be seen that the microhardness increases progressively with strain level. This was attributed on the one hand to the dislocation storage and the development of dislocation density, the re-orientation of cementite lamellae into the drawing direction and the reduction of interlamellar spacing. On the other hand to the refinement of grains size as is demonstrated previously in grain size curve in fig. 2a [START_REF] Fang | Microstructure and mechanical properties of cold-drawn pearlitic wires affect by inherited texture[END_REF][START_REF] Sahay | Pearlitic Steels: Wire Drawing[END_REF][START_REF] Zhang | Microstructure and strengthening mechanisms in cold-drawn pearlitic steel wire[END_REF]. Fig. 3 stress-strain curves of the drawn pearlitic wires at a different strain.

Strain level ε

Table3. Mechanical properties of the drawing wires with a different stage of strain level (Di), drawing strain (.

CMn

Texture Evolution

Figure 6 shows Major axis inverse pole figure EBSD coloring maps of the drawn steel wires. These coloring maps allowed us to display the texture evolution of wires under wire drawing. It can be seen that the rod state wire had almost equiaxed grains and random textures (Fig. 6a) leading to isotropic structure. As the drawing progresses, a <110> fiber texture of the ferrite phase begins to develop gradually with deformation in the drawing direction as shown in fig. 6b-c. This takes the structure of the wire to change from isotropic to anisotropic. Numerous studies [START_REF] Zidani | Deformation textures in wire drawn perlitic steel[END_REF][START_REF] Kumar | Progressive Changes in the Microstructure and Texture in Pearlitic Steel during Wire Drawing[END_REF][START_REF] Nebbar | Microstructural Evolutions and Mechanical Properties of Drawn Medium Carbon Steel Wire[END_REF][START_REF] Zelin | Microstructure evolution in pearlitic steels during wire drawing[END_REF][START_REF] Djimaoui | Study of microstructural and mechanical behavior of mild steel wires cold drawn at TREFISOUD[END_REF][START_REF] Zidani | Mechanical behavior and texture evolution study of medium carbon steel wires during industrial wire-drawing process[END_REF] have observed the same texture. For example, Guo et al. [START_REF] Guo | Microstructure and texture evolution in fully pearlitic steel during wire drawing[END_REF] in their study have investigated the texture evolution of drawn pearlitic steel wires in three regions including the middle, center, and surface of the wire. their results indicate the development of <110> fiber texture of ferrite phase in all of those different regions.

The texture evolution in the ferrite phase of the drawn pearlitic wires with different levels of strain was presented in terms of {110} inverse pole figure. Thus, the intensity of <110> fiber texture of the wire rod and the drawn wires was quantified and presented in fig. 7. It can be seen that for the first case when the strain level ε = 1.16, the max of the intensity of <110> fiber texture increased rapidly and passed from 2.49 to 8.27. This intensity of <110> fiber texture increased more to reach 12.62 at higher deformation when ε = 2.01 and became saturated. The same results were reported by Nebbar et al. [START_REF] Nebbar | Microstructural Evolutions and Mechanical Properties of Drawn Medium Carbon Steel Wire[END_REF] for drawn medium carbon steel wires but in their investigation the intensity of <110> fiber texture increased and reached a value of 5.2 and 6.6 for 43.6% and 60% deformation levels, respectively. Compared to our study, the intensity of <110> fiber texture was very remarkable for the same level of deformation.

Conclusion

Texture evolution and mechanical properties of cold drawn pearlitic steel wires are investigated in this paper. Based on the results, it can be concluded that :

-The results show that the wire rod had almost random textures resulting in isotropic structure.

As the drawing proceeds, a strong <110> // ND fiber texture of ferrite phase forms and begins to develop gradually with deformation leading to a transformation of structure from isotropic to anisotropic. In addition, the quantitative analysis confirmed the results of micro-textures and shows that the maximum intensity of <110> fibre texture increased more to reach 12.62 at higher deformation and became saturated.

-Experiences on drawn steel wires have shown that the wire drawing has a strong influence on these wires where tensile strength and 0,2% yield strength increased substantially with the strain level. While that reduced the elongation.

-Wire drawing induces the increase of dislocation density, reduction of interlamellar spacing and the refinement of grains size which leads to a strong hardening of the wires.

Fig. 1 Fig. 2 a

 12 Fig. 1 SEM micrographs of a longitudinal section of cold drawing steel specimens at different strain levels: a) ε = 0.00 (wire rod) b) ε = 2.01

Fig. 4

 4 Fig. 4 Mechanical properties evolution as a function of drawing strain.

Fig. 5

 5 Fig. 5 Elongation evolution of the drawn pearlitic wires during drawing.

Fig. 6 Fig. 7 {110}

 67 Fig. 6 Major axis inverse pole figure EBSD maps and IPF color-coded maps of the cold drawing pearlitic steel wires at different strain levels: a) ε = 0.00 wire rod b) ε = 1.16 c) ε = 2.01

  Chemical composition of high-carbon steel wire rod used in this work.

	Table1.
	Composition	C	Mn	Si	P	Cr	Ni	S	V	Cu	Mo	Co
	Wt%	0.89 0.63 0.32 0.013 0.31 0.024 0.012 0.004 0.011 0.002 0.006
			Specimens		𝐝 𝐢 [mm]		Strain level ε
			Wire rod		11			0.00
			Wire draw 1		6.15			1.16
			Wire draw 2		4.03			2.01

Acknowledgments

This work is supported by the Algerian Ministry of Higher Education and Scientific Research and included as a part of PRFU project. The authors are very grateful to Trefesoud company (The National Company of Wire Drawing and Manufacture of Welding Products, Algeria) for supplying steel wires used in this investigation.

The authors would like also to acknowledge the technical team of exploitation division of Trefesoud company and the Thin Films and Applications Physics Laboratory (LPCMA) for their assistance and their contribution to conducting some of the experiments.