Valérie Haurie
email: valerie.haurie@inserm.fr

Ludovic Ménard

Alexandra Nicou
email: alexandranicou@yahoo.fr

Christian Touriol
email: christian.touriol@inserm.fr

Philippe Metzler
email: metzlerphilippe@hotmail.com

Jérémy Fernandez
email: j.fernandez40@yahoo.fr

Danièle Taras
email: daniele.taras@inserm.fr

Patrick Lestienne
email: patrick.lestienne@inserm.fr

Charles Balabaud
email: charles.balabaud@chu-bordeaux.fr

Paulette Bioulac

Paulette Bioulac-Sage
email: paulette.bioulac-sage@gref.u-bordeaux2.fr

Hervé Prats
email: herve.prats@inserm.fr

Jessica Zucman-Rossi
email: jessica@inserm-u674.net

Jean Rosenbaum
email: jean.rosenbaum@gref.u-bordeaux2.fr

From

Adenosine triphosphatase pontin

Keywords: proteasome, translation, helicase, prognosis, ubiquitin proteasome, ubiquitin, translation, helicase, prognosis HCC, Hepatocellular carcinoma; AAA+, ATPases associated with various cellular activities

Hepatocellular carcinoma (HCC) is the main type of primary liver cancer. It is the fifth most common cancer worldwide and bears a very poor prognosis, mainly because of the lack of efficient therapy. Looking for new targets, we performed a comparative proteomic analysis between HCC and peri-tumoral liver, which led to the discovery of many deregulated proteins (1), including Reptin. Reptin overexpression was associated with a poor prognosis, and we demonstrated that Reptin was required for HCC cell viability and growth, and that Reptin overexpression conferred a greater ability to tumor cells for growth in xenografts (2).

Reptin (TIP48, TIP49b, RuvBL2, TAP54β, ECP-51) and Pontin (or TIP49, RuvBL1, TAP54α, ECP-54) are closely-related members of the AAA+ family of ATPases (ATPases Associated with various cellular Activities (3)) that show remarkable conservation from yeast to human. Due to their participation in several distinct multi-protein complexes (reviewed in (4,5)), they are involved in chromatin remodeling [START_REF] Jonsson | Rvb1p and Rvb2p are essential components of a chromatin remodeling complex that regulates transcription of over 5% of yeast genes[END_REF][START_REF] Ikura | Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis[END_REF][START_REF] Shen | A chromatin remodelling complex involved in transcription and DNA processing[END_REF], transcriptional regulation [START_REF] Jonsson | Rvb1p and Rvb2p are essential components of a chromatin remodeling complex that regulates transcription of over 5% of yeast genes[END_REF][START_REF] Kim | Transcriptional regulation of a metastasis suppressor gene by Tip60 and beta-catenin complexes[END_REF][START_REF] Bauer | Pontin52 and reptin52 function as antagonistic regulators of beta-catenin signalling activity[END_REF], DNA damage repair [START_REF] Ikura | Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis[END_REF][START_REF] Gospodinov | RAD51 foci formation in response to DNA damage is modulated by TIP49[END_REF], snoRNA biogenesis [START_REF] King | A well-connected and conserved nucleoplasmic helicase is required for production of box C/D and H/ACA snoRNAs and localization of snoRNP proteins[END_REF] and telomerase activity [START_REF] Venteicher | Identification of ATPases pontin and reptin as telomerase components essential for holoenzyme assembly[END_REF]. Because Reptin is usually associated with Pontin in complexes where they are present in stoichiometric amounts [START_REF] Ikura | Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis[END_REF][START_REF] Shen | A chromatin remodelling complex involved in transcription and DNA processing[END_REF][START_REF] Bauer | Pontin52 and reptin52 function as antagonistic regulators of beta-catenin signalling activity[END_REF][START_REF] Bauer | Pontin52, an interaction partner of beta-catenin, binds to the TATA box binding protein[END_REF][START_REF] Kanemaki | Molecular cloning of a rat 49-kDa TBP-interacting protein (TIP49) that is highly homologous to the bacterial RuvB[END_REF][START_REF] Wood | An ATPase/helicase complex is an essential cofactor for oncogenic transformation by c-Myc[END_REF][START_REF] Puri | Dodecameric structure and ATPase activity of the human TIP48/TIP49 complex[END_REF][START_REF] Torreira | Architecture of the pontin/reptin complex, essential in the assembly of several macromolecular complexes[END_REF][START_REF] Zhao | Navigating the chaperone network: an integrative map of physical and genetic interactions mediated by the hsp90 chaperone[END_REF], we studied Pontin expression and function in HCC. We found that Pontin was also up-regulated in HCC and unexpectedly that Pontin and Reptin expression was co-regulated via a novel post-translational mechanism.

Experimental procedures

Liver samples and real-time RT-PCR

Samples came from resected or explanted livers with HCC of patients treated in Bordeaux from 1992 to 2005. Fragments of fresh tumor and non-tumor liver tissues (taken at a distance of at least 2 cm from the tumor) were either snap-frozen in liquid nitrogen and stored at -80°C, or fixed with formalin and embedded in paraffin. 104 HCC samples (supporting Table HEP-09-0792 5 1) were used for real-time RT-PCR analysis. 18 non-tumor liver samples were used as a control group. RNA extraction and real-time RT-PCR were performed as described (2,[START_REF] Bioulac-Sage | Clinical, morphologic, and molecular features defining so-called telangiectatic focal nodular hyperplasias of the liver[END_REF].

Pre-developed sequence detection reagents specific for human RUVBL1 gene (Applied Biosystems, Courtaboeuf, France) were used as described (20) using the 2 -∆∆CT method [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF].

Gene expression results were normalized to internal control ribosomal 18S.

Immunohistochemistry

This was done as described (2) using a mouse monoclonal Pontin antibody [START_REF] Weiske | The histidine triad protein Hint1 interacts with Pontin and Reptin and inhibits TCF-beta-catenin-mediated transcription[END_REF] diluted to 1.6 µg/µl.

Transient transfection of small interfering RNA (siRNA)

We used two targeting siRNAs for each Reptin (R1 and R2 (2)) and Pontin mRNAs (P1 and P2, Supporting Table 2). Controls were either scrambled R2 and P2 sequences, or the GL2 siRNA targeting Firefly luciferase (MWG, Ebersberg, Germany). siRNAs were transfected at a concentration of 125nM with Lipofectamine (Invitrogen, Cergy Pontoise, France).

Cell proliferation assay and caspase 3 activity measurement

Cells were counted with a Coulter counter (Beckman Coulter, Villepinte, France) in duplicate wells. DNA synthesis was measured by the quantification of bromodeoxyuridine (BrdU) incorporation, and caspase 3 activity with a colorimetric assay (2).

Western blot

Cell extracts were prepared in RIPA buffer [START_REF] Neaud | Thrombin Upregulates Tissue Factor Pathway Inhibitor-2 Synthesis through a Cyclooxygenase-2dependent, Epidermal Growth Factor Receptor-independent Mechanism[END_REF]. We used Reptin, β-catenin (BD Biosciences, Pharmingen, Le Pont de Caix, France), FLAG-M2, β-actin (Sigma-Aldrich, Saint-Quentin Fallavier, France) mouse monoclonal, and Pontin rabbit polyclonal (ProteinTech, Chicago,

Polyribosome fractionation

KGL2 and KR2 cells lines stably expressing an shRNA targeting Firefly luciferase or Reptin, respectively, in a doxycycline-dependent manner (Supporting Methods), were cultured with or without doxycycline. Cycloheximide (100 µM) was added ten minutes before cell lysis.

Sucrose-gradient fractionation and polysome-associated RNA purification were as described [START_REF] Bastide | An upstream open reading frame within an IRES controls expression of a specific VEGF-A isoform[END_REF]. RNAs were analyzed by cDNA synthesis and PCR amplification.

Metabolic labeling and immunoprecipitation

KGL2 and KR2 cells were stably transduced with a lentiviral vector coding HA-Pontin resulting in KGL2-HAP and KR2-HAP cell lines (Supporting Methods). Similarly, KP2 cells expressing the P2 Pontin shRNA (Supporting Methods), were transduced with Flag-Reptin (2), resulting in KP2-FR cells. Cells were incubated in Methionine/Cysteine-free medium for 30 min before pulse labeling with 150 µCi/ml EXPRE 35 Cell extracts were diluted in lysis buffer without SDS and incubated for 2 hours at 4°C with monoclonal anti-HA-Agarose or anti-Flag M2 beads (Sigma-Aldrich). The beads were washed 5 times with lysis buffer and eluted with Laemmli sample buffer. Eluates were separated on a 10% SDS-PAGE. In some cases, Pontin was precipitated with a polyclonal anti-Pontin antibody.

Signals from radiolabeled protein bands were acquired in an Instant-Imager (Packard, Perkin-Elmer). Protein degradation rate is expressed as half-life (t 1/2 ), the time where 50% of the protein is degraded. The results are expressed as mean ± SEM of five to six independent determinations.

Statistical analysis

Statistical analyses were performed using the 2-tailed Student t test, or 1-way analysis of variance (ANOVA) when comparing multiple means. Statistical significance was set at a p value of less than 0.05. Correlations between mRNA levels of expression and qualitative variables were calculated with the non-parametric Kruskal-Wallis test with STATA software (Stata Corp., College Station, TX). The ages of patients and diameters of the tumors were partitioned with a median. For survival analysis, expression data were logarithm 10transformed and dichotomized according to the median. Survival comparisons were done by log-rank test. Survival curves were obtained by the Kaplan-Meier method. Multivariate analyses were performed using Cox model. All survival analyses were performed using STATA software 8.2.

Results

Pontin is overexpressed in human HCC

There was a 3.9-fold increase in Pontin transcripts in HCC as compared to non-tumor livers (Fig. 1A, p<0.0004). Pontin mRNA levels were highly correlated with those of Reptin mRNA (r 2 = 0.78, p < 0.0001, Fig. 1B). Immunohistochemistry confirmed the overexpression of Pontin in HCC with the same pattern as Reptin, and showed that, similar to Reptin (2), Pontin was partly localized to the cytoplasm of tumor cells (Fig. 1C-E). Pontin mRNA levels were correlated with features of poor prognosis : levels were higher in large tumors (> 60 mm, p = 0.026), in tumors with vascular embolism (p = 0.047), portal invasion (p = 0.008), or chromosomal instability (fractional allelic loss > 0.128, p = 0.02), in patients with early relapse following curative surgery (p = 0.001) or who died within 3 years of surgery (p = 0.001). A significant shorter disease-free survival in patients with high Pontin level was observed as compared to those with low level (Fig. 1F, p = 0.0022). Most importantly, using a Cox model multivariate analysis, high Pontin mRNA levels remained associated with shorter disease-free survival after adjustment for age, gender, tumor diameter and the presence of vascular embolism (Hazard Ratio = 3.9, 95% CI = [1.9-8.0], p=0.003).

Pontin silencing reduced cell proliferation and induced apoptosis

Pontin mRNA level was significantly reduced with P1 (70% ± 13%) and P2 (77% ± 12%) siRNAs (Fig. 2A). The siRNAs also greatly decreased Pontin protein expression (78.6% ± 6.9% with P1 and 87.7% ± 9.0% with P2) (Fig. 2B).

Starting from Day 3 after transfection, the growth of cells transfected with Pontin siRNAs strikingly decreased (Fig. 2C). This was associated with both reduced DNA synthesis,

Concomitant down-regulation of Reptin and Pontin

Because findings with Pontin silencing were reminiscent of those we obtained previously upon Reptin silencing, we evaluated Reptin expression after transfection of Pontin siRNAs.

Surprisingly, Reptin protein level was markedly reduced after transfection of P1 or P2 compared to control siRNA. The reciprocal observation was done in cells transfected with the Reptin-specific R1 and R2 siRNAs where Pontin protein level was also decreased (Fig. 3A-B). Because of the significant homology between Reptin and Pontin, we performed extensive control experiments that allowed excluding cross-reactivity of antibodies (Supporting Fig. 1).

To rule out off-target effects of siRNAs, we used a rescue strategy. Cell lines resistant to siRNAs were established by transduction of lentiviral vectors bearing either HA-Pontin or Flag-Reptin cDNA in which silent mutations have been introduced in the sequence targeted by the P2 or R2 siRNAs (Supporting Fig. 2). Reptin protein expression was no more decreased by P2 siRNA in P2-resistant cells, whereas it was reduced as expected upon transfection of P1 siRNA (Fig. 3C). Similarly, Pontin expression was not affected in cells expressing FLAG-Reptin resistant to the R2 siRNA (Fig. 3D). Altogether, these results showed that the down-regulation of Pontin was a direct consequence of the silencing of Reptin and reciprocally.

We then asked if our findings were specific of HuH7 cells. We examined the consequences of Reptin or Pontin silencing in another human liver cell line (HepG2), in human breast cancer cells (MCF7), in human prostatic cancer cells (LNCap) and in Hela cells. We also tested the HEP-09-0792 10 co-extinction of Pontin after transfection of the R2 siRNA in mouse (Hepa 1-6) or rat hepatoma cells (FAO). In every case, we observed a concomitant reduction of Reptin and Pontin protein levels (Supporting Fig. 3). These results argued in favor of a general rather than a cell-specific process for regulation of Pontin and Reptin expression.

Reptin silencing does not decrease Pontin mRNA level (and reciprocally)

Reptin and Pontin interact with transcription factors and bind to gene promoters (reviewed in ( 4)). We thus hypothesized that they could regulate reciprocally their expression through a transcriptional mechanism. However, transfection of Pontin siRNAs did not alter Reptin mRNA levels, whereas as expected, Pontin mRNA decreased. Reciprocally, Reptin, but not Pontin mRNA levels were efficiently silenced with Reptin siRNAs (Fig. 4A-B).

As an additional control, we tested whether Reptin produced from an mRNA devoid of its natural regulatory sequences would also be depleted upon transfection of a Pontin siRNA. We used HuH7 cells stably expressing N-term FLAG-tagged Reptin from the viral MND promoter (2). We found that the level of both endogenous and Flag-tagged Reptin was decreased after transfection of Pontin specific siRNAs (Supporting Fig. 4A). We obtained similar results with HeLa cells expressing FLAG/HA-tagged Reptin or Pontin from the CMV promoter (Supporting Fig. 4B).

Altogether, these experiments allow concluding that the reduction of Reptin or Pontin protein levels upon silencing of the respective binding partner is not related to a transcriptional effect, and thus occurs by a post-transcriptional mechanism. Given the pleiotropic role of Reptin and Pontin, we hypothesized that they might regulate their partner's translation. This was tested using polyribosome fractionation and RT-PCR analysis in HuH7 cell lines stably expressing shRNAs targeting either Firefly luciferase (KGL2) or Reptin (KR2), in a doxycycline-dependent manner (Supporting Fig. 5). The polysomal distribution of Pontin mRNA was analyzed after 4 days of culture with or without doxycycline (Fig. 5A-B). In KGL2 cells, Reptin and Pontin transcripts exhibited a similar profile and were found all along the gradient, with an enhancement in the heaviest fractions, associated with polyribosomes. As expected, in doxycycline-treated KR2 cells, Reptin mRNA became barely detectable, whereas the profile of Pontin mRNA was unchanged as compared to that in KGL2 cells, and its distribution over the fractions was similar to the profile of GAPDH mRNA. When cells were incubated with puromycin for one hour prior to extraction, the profile of Pontin and GAPDH mRNAs shifted from heavy fractions to the top of the gradient (Fig. 5B). Because puromycin is a polypeptide chain terminator that causes premature termination of translation, our results demonstrate a genuine association of Pontin mRNA with polyribosomes, even in conditions where Reptin is depleted.

Reptin silencing does not alter Pontin mRNA translation (and reciprocally)

To make sure that Pontin mRNA was indeed actively translated, we performed metabolic labeling after 4 days of culture with doxycycline using cells stably expressing HA-Pontin (KGL2-HAP and KR2-HAP). After a 15 min pulse of radiolabeled methionine/cysteine, the level of 35 S incorporation into newly synthesized proteins was not different between KGL2-HAP and KR2-HAP cells (78.8% ± 4.3% vs 71.6 ± 6.8%, respectively), suggesting no gross alteration in overall protein biosynthesis in Reptin silencing conditions. After immunoprecipitation with anti-HA antibodies, the label accumulated in a 55-kDa band confirmed to be the full-length HA-Pontin polypeptide since it was reactive with an antibody against Pontin (Fig. 5C). When Reptin shRNA expression was induced in KR2-HAP cells, the

Reptin silencing induces Pontin destabilization

Because Pontin and Reptin form a stoichiometric complex, we hypothesized that downregulation of one of the two partners could disturb this stoichiometry and lead to posttranslational destabilization of the binding partner. We thus measured Pontin half-life using metabolic labeling-chase experiments. Newly synthesized HA-Pontin was less stable upon Reptin silencing as shown by a significantly reduced half-life as compared to control (t 1/2 = 66.6 ± 7.6 vs 118 ± 21 min, p = 0.002) (Fig. 6A,6C). Because the proteasome is the major intracellular proteolytic machinery in higher eukaryotic cells, we tested its involvement using specific inhibitors. Fig. 6B-C shows that MG132 was able to prolong the half-life of HA-Pontin in both Reptin-repleted and depleted conditions. The effect of Reptin depletion was specific since it did not alter the half-life of calnexin tested as a control (Fig. 6D).

We then asked if proteasome inhibitors could restore Pontin steady-state level in Reptindepleted cells. We performed many experiments using a variety of experimental settings. For instance, Fig. 7A shows an experiment where, three days after induction of Reptin shRNA expression, HuH7 cells were treated with MG132 or epoxomycin for 4h. This did not modify the co-depletion although effective inhibition of the proteasome was confirmed by the accumulation of higher molecular weight forms of β-catenin, a proteasome target [START_REF] Aberle | beta-catenin is a target for the ubiquitin-proteasome pathway[END_REF].

Similar results were seen when using other MG132 concentrations, duration of treatment, or with other inhibitors such as clasto-Lactacystin-β-lactone (not shown). We hypothesized that if the stabilizing effect of proteasome inhibition was restricted to newly synthesized protein chains, our experiments might not be sensitive enough to detect an accumulation of proteins within the short time course of the experiments. We thus repeated them using HuH7 cells that stably express HA-Pontin. These cells have high levels of Pontin mRNA (not shown), and interestingly a higher rate of Pontin translation than control cells (Fig. 7B). They do however overexpress very little Pontin at the protein level, reminiscent of our previous results with Flag-Reptin (2) (Fig. 7B). When these cells were treated with MG132 for 4 hours, we could detect a significant increase in Pontin protein levels both in basal conditions and when Reptin was previously depleted upon shRNA induction with doxycycline (Fig. 7C-D). On the other hand, the low level of Reptin consecutive to R2 shRNA induction in doxycycline-treated cells was not increased by MG132. Similar results were obtained when the proteasome was inhibited with 2.5µM epoxomycin (not shown).

Altogether, these results suggested that the restoration seen with proteasome inhibition was restricted to the pool of freshly translated protein. This hypothesis was tested in experiments where, following Reptin depletion, proteasome inhibition with MG1342 or epoxomycin was done in the absence or in the presence of cycloheximide that blocks new protein synthesis. As shown in Fig. 7E-F Since in most cases proteins are targeted to the proteasome following poly-ubiquitylation, we examined the stability of newly synthesized Pontin in the presence of the ubiquitin activating enzyme inhibitor UBEI-41 (Biogenova). We found indeed that newly synthesized Pontin stability was enhanced in cells with Reptin depletion when they were treated with UBEI-41 (Fig. 8A). We also performed the reverse experiment. We first showed that the stability of newly synthesized Reptin was reduced upon Pontin silencing (Fig. 8D, compare lanes 2 and 6), an effect reverted in great part by proteasome inhibition (Fig. 8D). Furthermore, Reptin stability in Pontin-depleted cells was also largely restored following inhibition of ubiquitin activating enzyme (Fig. 8D). These data proved that an ubiquitylation step was involved in the destabilization of Pontin and Reptin in this setting.

We next attempted to detect whether Pontin and Reptin underwent polyubiquitylation upon silencing of their partner. Because endogenous ubiquitin might be limiting, and in order to maximize the sensitivity of detection, we transduced KR2 cells with adenoviral vectors (kindly provided by H. Wodrich, Bordeaux) expressing either HA-ubiquitin and GFP, or GFP alone. After 3 days with or without Dox, the proteasome was inhibited with epoxomycin in order to allow accumulation of ubiquitylated proteins, and ubiquitin was precipitated using the HA tag. Western blot with a Pontin antibody failed to reveal ubiquitylated Pontin whereas ubiquitylated β-catenin was indeed detected (Supporting Fig. 6A). The same results were obtained when looking for Reptin ubiquitylation in KP2-FR cells (Supporting Fig. 6B).

Discussion

We show that Pontin is overexpressed in a large series of human HCC. High levels of Pontin mRNA were associated with features of poor prognosis. Using a multivariate analysis, we even found that Pontin level was an independent factor of poor prognosis when taking into Surprisingly, silencing experiments led to a similar co-depletion of Pontin and Reptin, as recently found in HeLa cells [START_REF] Venteicher | Identification of ATPases pontin and reptin as telomerase components essential for holoenzyme assembly[END_REF]. Our results were extended to a series of human cells from liver and non-liver origin and to other species, suggesting that co-depletion of Reptin and Pontin is a general finding. We demonstrate that Pontin and Reptin co-depletion is linked to a post-translational regulation since no changes in mRNA levels nor in translation were observed following silencing. On the other hand, we show that the stability of newly synthesized Pontin is reduced when Reptin is previously depleted, and reciprocally. This defect can be reverted when using a proteasome inhibitor. In addition, we show that proteasome inhibition is able to increase Pontin steady-state level whether Reptin levels are high or low. Proteasome inhibition had on the other hand no effect on the residual low levels of Reptin consecutive to RNAi. Together with our results with cycloheximide, this suggests that the restoration of Pontin levels due to proteasome inhibition is mainly the consequence of stabilization of newly translated Pontin, whereas no effect is seen for Reptin which translation is impaired because of RNAi. We suggest that Pontin and Reptin heteromers are formed immediately upon completion of translation, likely co-translationnally. In that case, in the absence of its partner, newly translated Pontin or Reptin cannot be correctly folded and is targeted for proteasomal degradation. This is also in agreement with our data showing that Reptin and Pontin depletion follow identical kinetics upon use of any siRNA (Supporting Fig. 7). It is also possible that Pontin and Reptin act as chaperones for each other. A chaperone role can be inferred from their presence in a HSP90 complex conserved in human and yeast [START_REF] Te | Novel subunits of the mammalian Hsp90 signal transduction chaperone[END_REF][START_REF] Zhao | Molecular chaperone Hsp90 stabilizes Pih1/Nop17 to maintain R2TP complex activity that regulates snoRNA accumulation[END_REF], their role in the assembly of the Ino80 complex in yeast [START_REF] Jonsson | Rvb1p/Rvb2p recruit Arp5p and assemble a functional Ino80 chromatin remodeling complex[END_REF], and is coherent with the fact that many other members of the AAA+ family are indeed bona fide chaperones (3). In most cases, proteins undergo proteasome degradation following prior polyubiquitylation.

Polyubiquitylation involves an ordered set of reactions beginning with activation of ubiquitin by the ubiquitin-activating enzyme E1 [START_REF] Glickman | The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction[END_REF]. We thus used an E1 inhibitor and found that the stability of both Pontin and Reptin was enhanced. We were however unable to directly detect polyubiquitylation of any of them even when ubiquitin was greatly overexpressed and proteasome inhibited. It may be that the sensitivity of ubiquitylation detection is still insufficient or that there is an intermediary protein that is ubiquitylated and regulates Pontin and Reptin turnover [START_REF] Jariel-Encontre | Ubiquitin-independent degradation of proteins by the proteasome[END_REF]. Further experiments are needed to clarify this point.

Besides the co-depletion, results from our study also shed light on our previous observation that transduction of cells with a lentiviral vector coding Flag-tagged Reptin induced very little protein overexpression despite highly increased mRNA levels (2). We obtained similar data here with a vector coding HA-Pontin, and found that inhibition of proteasome enhanced the stability of newly synthesized Pontin and allowed a higher accumulation of Pontin (Fig. 7C-D). This gives support to the hypothesis that the lack of high-level protein expression is explained by a degradation of the newly translated protein that cannot find its binding partner.

A co-depletion of proteins engaged in functional complexes has been already shown for instance in the case of the Ku70-Ku80 heterodimer [START_REF] Nussenzweig | Requirement for Ku80 in growth and immunoglobulin V(D)J recombination[END_REF] or the γ-secretase complex [START_REF] Edbauer | Presenilin and nicastrin regulate each other and determine amyloid beta-peptide production via complex formation[END_REF]. However, in no case were full mechanistic data provided. In addition, our findings are unique in that we show that destabilization of the protein partner intervenes at an early posttranslational stage. From our findings, one would expect that the depletion of either protein would result in the same cellular phenotype. This is indeed what was observed in our experiments when comparing depletion of Reptin (2) and Pontin (this study) on cell growth and apoptosis, and also by others on the formation of RAD51 nuclear foci after DNA damage in prostate cancer cells [START_REF] Gospodinov | RAD51 foci formation in response to DNA damage is modulated by TIP49[END_REF]. In the yeast, genome-wide microarray analysis after acute deletion of either protein showed a striking correlation between the genes regulated by each protein [START_REF] Jonsson | Rvb1p and Rvb2p are essential components of a chromatin remodeling complex that regulates transcription of over 5% of yeast genes[END_REF]. More generally, knockdown, inactivation or deletion of either gene in Drosophila [START_REF] Bauer | Pontin52 and reptin52 function as antagonistic regulators of beta-catenin signalling activity[END_REF] or yeast [START_REF] Kanemaki | TIP49b, a new RuvB-like DNA helicase, is included in a complex together with another RuvB-like DNA helicase, TIP49a[END_REF] resulted in a similarly lethal phenotype. These data have been interpreted as Pontin and Reptin having non-redundant functions. We suggest that they could be re-interpreted on the basis of the co-depletion of both proteins. Still, it is likely that Pontin and Reptin have some unique functions as suggested by several observations. Indeed, whereas Pontin depletion with siRNAs led to spindle defects in Drosophila S2 and HeLa cells, Reptin siRNAs had no effect [START_REF] Ducat | Regulation of Microtubule Assembly and Organization in Mitosis by the AAA+ ATPase Pontin[END_REF]. Pontin and Reptin also antagonistically modulate β-catenin transcriptional activity [START_REF] Bauer | Pontin52 and reptin52 function as antagonistic regulators of beta-catenin signalling activity[END_REF][START_REF] Bauer | Pontin52, an interaction partner of beta-catenin, binds to the TATA box binding protein[END_REF][START_REF] Wood | An ATPase/helicase complex is an essential cofactor for oncogenic transformation by c-Myc[END_REF][START_REF] Weiske | The histidine triad protein Hint1 interacts with Pontin and Reptin and inhibits TCF-beta-catenin-mediated transcription[END_REF], likely because they are incorporated into different complexes and differentially recruited onto target promoters [START_REF] Kim | Transcriptional regulation of a metastasis suppressor gene by Tip60 and beta-catenin complexes[END_REF][START_REF] Diop | Reptin and Pontin function antagonistically with PcG and TrxG complexes to mediate Hox gene control[END_REF]. These apparently unique functions may be due to a small pool of Pontin and Reptin not engaged in heteromeric complexes but present as monomers of homo-oligomers. Although structural studies suggest mostly hetero-complex formation [START_REF] Puri | Dodecameric structure and ATPase activity of the human TIP48/TIP49 complex[END_REF][START_REF] Torreira | Architecture of the pontin/reptin complex, essential in the assembly of several macromolecular complexes[END_REF], they do not exclude the possibility of other forms [START_REF] Torreira | Architecture of the pontin/reptin complex, essential in the assembly of several macromolecular complexes[END_REF]. In summary, Pontin, like Reptin, is overexpressed in HCC and is required for HCC growth and viability. We demonstrate that the expression of both proteins is strictly co-regulated by a post-translational mechanism involving the proteasomal degradation of newly synthesized proteins. We also found that Pontin and Reptin expression are highly correlated at the mRNA level in HCC thus suggesting some additional co-regulation at the mRNA level. Altogether, Louis, MO). This vector drives the expression of the transcriptional repressor tTR-KRAB and of the marker dsRED. Then, HuH7 cells expressing tTR-KRAB were selected through dsRED expression in a cell sorter (FACS Aria, BD Biosciences, San Jose, CA). Secondly, HuH7 expressing tTR-KRAB were transduced with PLVTHM-GL2 or PLVTHM-R2 at a multiplicity of infection of 15. The resulting cell lines were named KGL2 and KR2. The transduction efficiency was checked through testing for EGFP expression by flow cytometry after three days of cultivation in medium containing 0.02 µg/ml doxycycline. The KP2 cell line expressing the Pontin P2 shRNA was constructed similarly using the following oligonucleotides : P2 sense, 5'-cgcgtCCCCGGTGAAGTCACAGAGCTAAttcaaga gaTTAGCTCTGTGACTTCACCTTTTTGGAAat-3' ; P2 5'-antisense, cgatTTCCAAAAAG GTGAAGTCACAGAGCTAAtctcttgaaTTAGCTCTGTGACTTCACCGGGGa-3'.

Construction of cell lines expressing HA-tagged Pontin

N-term HA-tagged Pontin cDNA was amplified by PCR from CBS-Tip49 (4) and subcloned in pGEM-Teasy plasmid (Promega). The cDNA was sequenced and cloned downstream of the MND (Myeloproliferative sarcoma virus enhancer, Negative control region Deleted, d1587rev primer-binding site substituted) promoter in a lentiviral vector (1). Finally, KGL2 and KR2 cell lines were transduced with this vector at a multiplicity of infection of 2. The resulting cell lines were named KGL2-HAP and KR2-HAP.

Construction of cell lines expressing Reptin or Pontin resistant to siRNA

Human N-term FLAG-tagged Reptin and N-term HA-tagged Pontin cDNAs were amplified by PCR from CBS-Tip48 and CBS-Tip49(4) and subcloned in pGEM-Teasy plasmid. Silent mutations were introduced by in vitro site-directed mutagenesis using the Stratagen QuikChange® II XL Site-Directed Mutagenesis Kit in the regions that are targeted by siRNAs (Supporting Figure 2). The inserts were sequenced to verify that selected clones contain the desired mutations and cloned downstream of the MND promoter in a lentiviral vector (1).

RNA isolation from cells, reverse transcription, real-time PCR

Total RNA was extracted with Trizol Reagent. One µg RNA was reverse-transcribed with Superscript III (Invitrogen). Real-time PCR was performed in 25µl with SybrGreen Supermix (BioRad, Marnes-la-Coquette, France). The sequences of primers used are in Supporting Table 2. Five µl of a 50-fold dilution of cDNA were used as template.

  . Primary antibodies were detected by horseradish peroxidase conjugated, or infrared dye-labeled secondary antibodies (LI-COR, Lincoln, NE). Detection was achieved with the ECL kit (GE Healthcare, Saclay, France) or the Odyssey IR imaging system (LI-COR), respectively.

  S 35 S Protein Labeling Mix (Perkin Elmer, Courtaboeuf, France) for 15 min at 37°C. Cells were washed then scraped in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 4 mM EDTA, 1% Triton, 1% SDS) supplemented with protease inhibitor cocktail (Roche, Meylan, France). The amount of radiolabeled TCA-precipitated material was measured by scintillation counting.For pulse-chase experiments, cells were labeled with 100 µCi/mL EXPRE 35 S 35 S Protein Labeling Mix for 1h, washed with medium supplemented with 2 mM cysteine/methionine, various times in this chase medium supplemented with the indicated agents before harvesting.

  BrdU incorporation, and increased apoptosis, demonstrated by an increased caspase 3 activity (Fig.2D).

  labeled HA-Pontin immunoprecipitated after a 15 min pulse was 98% of the control. This experiment showed that the decrease in HA-Pontin expression in cells expressing Reptin shRNA was not the consequence of a defective translation. Therefore, the association of Pontin mRNA with polyribosomes and the completion of HA-Pontin translation argued in favor of a post-translational regulation that explains the co-depletion of Pontin and Reptin. In reciprocal experiments, we also found that Pontin silencing did not alter Reptin translation, although Reptin silencing with R1 siRNA did reduce Reptin translation as expected (Fig.5D).

  , cycloheximide treatment prevented the restoration of Pontin levels in these conditions.

  prognosis factors including the presence of vascular embolism. In addition, in vitro experiments showed that Pontin was required for HCC cell growth and viability. All these findings, also including the cytoplasmic localization of Pontin in tumor cells, are highly reminiscent of those that we obtained with Reptin (2) and are in keeping with the known association of Pontin with Reptin into functional complexes(4, 5).

Figure 1 .

 1 Figure legends

Figure 2 .

 2 Figure 2. Efficiency and effects of silencing of Pontin by siRNAs in HuH7 cells

Figure 3 .

 3 Figure 3. Silencing of Reptin or Pontin by specific siRNAs leads to down-regulation of

Figure 4 .

 4 Figure 4. Expression of Reptin and Pontin transcripts following silencing

Figure 5 .

 5 Figure 5. Distribution of Reptin and Pontin mRNA in polysomes, and translation of

Figure 6 .

 6 Figure 6. Half-life of Pontin

Figure 7 .

 7 Figure 7. Effect of proteasome inhibitors on the decrease in Pontin levels upon Reptin

  Fifteen µg total RNA was separated by electrophoresis into a formaldehyde-agarose gel (1.2% agarose). At the end of migration, RNAs were transferred by capillarity and UV-cross linked on a HyBond N+ nylon membrane.The probes used were the full-length Pontin and Reptin cDNA, purified after restriction digestion of plasmids CBF-Tip48 and CBF-Tip49 (4). The probes were radio-labeled using 5ng of template DNA with α 32 P-dCTP by random priming labeling according to the supplier's instruction (GE Healthcare Ready-to-Go DNA Labelling Beads). Hybridization was performed for 16 hours at 42°C in Expresshyb™ Hybridization solution (Clontech, BD).The signals were acquired in an Instant-Imager.

SupportingFig. 7 .

 7 Kinetics of co-depletion(A) HuH7 cells were transfected with the indicated siRNAs and protein expression was monitored by Western blot at various time points. (B) KR2 cells were grown in medium supplemented or not with doxyxcycline and Pontin or Reptin protein expression was monitored by Western blot at various time points.

  Supporting Table1. Patients characteristics and histology of the surrounding liver

Acknowledgments

We thank Pierre Laurent-Puig (Paris, France) for help with statistical analysis, Otmar Huber (Berlin, Germany) for antibodies, Michael Cole (Lebanon, NH, USA) and Irina Tsaneva (London, UK) for plasmids, Harald Wodrich (Bordeaux, France) for ubiquitin tools and know-how, and Marc Piechaczyk (Montpellier, France) for helpful discussions.

Grant support : The work was supported by grants from Agence Nationale pour la Recherche sur le SIDA et les Hépatites Virales, Institut National du Cancer (PL06_117), Association pour la Recherche sur le Cancer (4026), Ligue Nationale Contre le Cancer and Conseil Régional d'Aquitaine. VH was the recipient of a fellowship from Agence Nationale pour la Recherche sur le SIDA et les Hépatites Virales, and AN from Institut National du Cancer.

Pontin. There was a 4.9 fold increase in newly synthesized Pontin in cells expressing HA-Pontin as compared to control cells. On the right side, Western blot for Pontin. Pontin level was increased by only 1.2 fold in KR2 HAP cells as compared to control cells. (C) After 3 days of culture with or without doxycycline, cells were treated for 4h with [START_REF] Aberle | beta-catenin is a target for the ubiquitin-proteasome pathway[END_REF]

Supporting information

Supporting methods

Cell culture

HuH7 cells were cultured in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, non essential amino acids, L-glutamine, and penicillin/streptomycin in 5% CO 2 -containing atmosphere at 37°C.

Construction of cell lines with a conditional expression of Reptin or Pontin shRNA

The Reptin siRNA sequence chosen was the R2 sequence previously described (1) Finally, HuH7 cells conditionally expressing GL2-or R2-shRNA were obtained as follows.

First, HuH7 cells were transduced with the PLVtTR/KRAB-Red lentiviral vector (2) at a multiplicity of infection of 5 in Dulbecco/Vogt modified Eagle's minimal essential medium (DMEM)/10% fetal bovine serum (FBS) medium with 8 µg/ml protamine sulfate (Sigma, St.

Supporting Figure legends


Guillem López De Egea

Aida González-Díaz

Gérard Guédon

Julie Lao

Dàmaris Berbel

Antonio Casabella

José María Marimón

Emilia Cercenado

Lucía Fernández-Delgado

Hélène Chiapello

Manuela Oliveira

Elisabete Silva

Aida Gonzálezdía

Gérard Guédo

Dàmaris Berb

José María Marim

Thoma Lacroi

María Ángeles Domínguez

Nathalie Leblond-Bourget

Carmen Ardanuy
email: c.ardanuy@bellvitgehospital.cat

A New Integrative and Mobilizable Element Is a Major Contributor to Tetracycline Resistance in Streptococcus dysgalactiae subsp. equisimilis

Keywords: Streptococcus dysgalactiae subsp. equisimilis, tet(M), IME, ICE, mobile genetic elements

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Streptococcus dysgalactiae subsp. equisimilis (SDSE) are beta-hemolytic streptococci that possess cell wall carbohydrate serogroup antigens C and G and sometimes A or L. SDSE are present in the microbiota of the respiratory, gastrointestinal, and female genital tract and the skin of humans [START_REF] Vandamme | Taxonomic Study of Lancefield Streptococcal Groups C, G, and L (Streptococcus dysgalactiae) and Proposal of S. dysgalactiae subsp. equisimilis subsp[END_REF][START_REF] Efstratiou | Pyogenic Streptococci of Lancefield Groups C and G as Pathogens in Man[END_REF]. It usually causes mild to severe infections in humans as Streptococcus pyogenes, ranging from pharyngitis and skin infections to necrotizing fasciitis and streptococcal toxic shock-like syndrome [START_REF] Efstratiou | Pyogenic Streptococci of Lancefield Groups C and G as Pathogens in Man[END_REF][START_REF] Brandt | Human Infections Due to Streptococcus dysgalactiae subspecies equisimilis[END_REF]. These two species are phylogenetically closely related and share many virulence factors, such as the M-protein, involved in the adhesion and immune system evasion, and used for epidemiological typing [START_REF] Jensen | Delineation of Streptococcus dysgalactiae, Its Subspecies, and Its Clinical and Phylogenetic Relationship to Streptococcus pyogenes[END_REF]. In recent years, two important facts related to SDSE have been described: first, the global emergence of invasive disease due to SDSE [START_REF] Lambertsen | Nationwide Laboratory-Based Surveillance of Invasive Beta-Haemolytic Streptococci in Denmark from 2005 to 2011[END_REF][START_REF] Anonymous | Voluntary Surveillance of Pyogenic and Non-Pyogenic Streptococcal Bacteraemia in England, Wales and Northern Ireland: 2020[END_REF][START_REF] Wajima | Molecular Characterization of Invasive Streptococcus dysgalactiae subsp. equisimilis, Japan[END_REF][START_REF] Rantala | Clinical Presentations and Epidemiology of β-Haemolytic Streptococcal Bacteraemia: A Population-Based Study[END_REF] and second, the description of the horizontal DNA transfer from SDSE to S. pyogenes, such as pbp2X gene, leading S. pyogenes to have a decreased susceptibility to beta-lactams [START_REF] Beres | Integrative Reverse Genetic Analysis Identifies Polymorphisms Contributing to Decreased Antimicrobial Agent Susceptibility in Streptococcus pyogenes[END_REF].

Tetracycline resistance can be mediated by three main mechanisms: active efflux pumps, ribosomal protection, and enzymatic inactivation. Within the genus Streptococcus, ribosomal protection is the most important mechanism, and the tet(M) gene the most frequent. This gene is mostly found in the integrative and conjugative elements (ICEs), mainly of the Tn916-family, which can spread through horizontal transmission by conjugation [START_REF] Chopra | Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance[END_REF]. In fact, having all the requirements for self-transmission is a hallmark of ICEs. On the other hand, integrative and mobilizable elements (IMEs) are elements that encode their own excision and integration, possess only some sequence and genes involved in conjugation, and hijack the conjugation apparatus of conjugative plasmids or ICEs to promote their own transfer [START_REF] Guédon | The Obscure World of Integrative and Mobilizable Elements, Highly Widespread Elements That Pirate Bacterial Conjugative Systems[END_REF]. Despite the fact that IMEs' prevalence is still underestimated, many of them encode resistance to antibiotics and altogether confer resistance to a large variety of antibiotics [START_REF] Guédon | The Obscure World of Integrative and Mobilizable Elements, Highly Widespread Elements That Pirate Bacterial Conjugative Systems[END_REF]. Considering resistance to tetracycline, several IMEs from Streptococcus suis were found to carry the tet(O) gene and one a tet(40) gene [START_REF] Libante | Chromosomal Conjugative and Mobilizable Elements in Streptococcus suis: Major Actors in the Spreading of Antimicrobial Resistance and Bacteriocin Synthesis Genes[END_REF][START_REF] Liang | Genomic and Pathogenic Investigations of Streptococcus suis Serotype 7 Population Derived from a Human Patient and Pigs[END_REF][START_REF] Wang | Characterization of Integrative and Conjugative Elements Carrying Antibiotic Resistance Genes of Streptococcus suis Isolated in China[END_REF]. However, no IME carrying tet(M) had ever been identified in Firmicutes. During a surveillance study of tetracycline resistance in SDSE we found a new IME carrying the tet(M) gene. Hence, the aim of the present study was to describe this new element and to analyze the epidemiology of tetracycline resistance in SDSE.

Results and Discussion

Out of a total of 84 SDSE isolates causing invasive disease (iSDSE), 24 (28.5%) were resistant to tetracycline. In order to analyze the trends in the iSDSE over the study period, we analyzed data from the Hospital Universitari de Bellvitge (HUB) covering the whole period. Even though the number of isolates collected per year was low, tetracycline resistance rates remained stable among the iSDSE collected from HUB from 37.5% (3/8) in 2012-2015 to 30.2% (16/53) in 2016-2019.

The genome analysis showed that tetracycline resistance was associated with the presence of the tet(M) (n = 22), tet(W) (n = 1), or tet(L) plus tet(W) (n = 1) genes. This gene distribution among the iSDSE agreed with previous results from other regions [START_REF] Lu | Molecular Characterization and Antibiotic Resistance of Clinical Streptococcus dysgalactiae subsp. equisimilis in Beijing, China[END_REF][START_REF] Rojo-Bezares | Streptococcus dysgalactiae subsp. equisimilis from Invasive and Non-Invasive Infections in Spain: Combining Epidemiology, Molecular Characterization, and Genetic Diversity[END_REF]. Nevertheless, exploring the genetic environment of the tet(M) gene, we found that it was carried by two different types of elements transferable by conjugation. In ten isolates, the tet(M) gene was carried by an ICE belonging to the Tn916-family [Tn916 (n = 9) and Tn3872 (n = 1, also having the erm(B) gene)], as previously described. On the other hand, 12 isolates had the tet(M) gene in a new IME (IME_SDSE_HUB-4529; Figures 1 and2) that was identical (100% nucleotide identity) in all 12 isolates. The IME_SDSE_HUB-4529 encodes a tyrosine integrase and a relaxase of the MOBT superfamily. All of them are integrated in the same site located in an intergenic region between the genes encoding for the alpha subunit of ribonucleotide reductase of class Ib (aerobic) and the Na + -dependent branched-chain amino acid transporter (Figure 2). A BLASTN search identified identical IMEs (100% identity) in the Enterococcus faecium (Acc. num AP019408.1) and Clostridioides difficile (Acc. num CP016102.1) genomes. The insertion sites of the element in the three species were different suggesting that the integrase is not site-specific. This fact together with the 100% identity of the IMEs and with the clustering of the strains on the phylogenetic tree suggests the clonality of the 12 strains and a recent acquisition of the IME by a common ancestor to these strains. A further BLASTN search identified similar structures in other Gram-positive bacteria (E. faecalis, C. innocuum, and Catenibacterium sp.) commonly found in the gut microbiota with an identity above 95% (Supplementary Figure S1). The presence of IME structures in iSDSE with a putative origin from other bacterial species is a cause for concern, especially for E. faecium which is known to be associated with vancomycin and multidrug resistance. In fact, transfer of the van(A) gene from enterococcal species to S. aureus has already been described [START_REF] Mcguinness | Vancomycin Resistance in Staphylococcus aureus[END_REF]. The chance of DNA exchanges is more plausible in the current scenario, with an increasing frequency of SDSE which is a cause of human infection.

that the integrase is not site-specific. This fact together with the 100% identity of the IMEs and with the clustering of the strains on the phylogenetic tree suggests the clonality of the 12 strains and a recent acquisition of the IME by a common ancestor to these strains. A further BLASTN search identified similar structures in other Gram-positive bacteria (E. faecalis, C. innocuum, and Catenibacterium sp.) commonly found in the gut microbiota with an identity above 95% (Supplementary Figure S1). The presence of IME structures in iSDSE with a putative origin from other bacterial species is a cause for concern, especially for E. faecium which is known to be associated with vancomycin and multidrug resistance. In fact, transfer of the van(A) gene from enterococcal species to S. aureus has already been described [START_REF] Mcguinness | Vancomycin Resistance in Staphylococcus aureus[END_REF]. The chance of DNA exchanges is more plausible in the current scenario, with an increasing frequency of SDSE which is a cause of human infection. Aside from the elements carrying tetracycline resistance, most of the isolates also had other resistance mechanisms carried by other elements (Figure 1). Among isolates carrying Tn916, one strain had the erm(T) gene in a non-self-transmissible plasmid and the dfr(F) in a different IME; two isolates had the Tn916 inserted into a larger ICE belonging to the Tn1549-family that also had erm(TR); a single isolate had the dfr(F) in a different IME; one isolate had the macrolide efflux genetic assembly (MEGA) element carrying the mef (E) gene; and finally, one isolate also had erm(B) located near an insertion sequence. On the other hand, three out of 12 isolates carrying the IME_SDSE_HUB-4529 also had the erm(TR) gene located in a Tn1549-family of ICE.

Table 1 shows the antibiotic susceptibility of the tetracycline-resistant SDSE. Betalactams (penicillin and cefotaxime), vancomycin, and linezolid were active against all the strains. In fact, beta-lactams are the first-line treatment of SDSE infections, and linezolid could be used as an alternative treatment in case of allergy. Regarding levofloxacin, all strains were "susceptible, increased exposure" following the EUCAST criteria. Nevertheless, one strain had a D83A substitution in ParC and had a MIC of levofloxacin of 2 mg/L which, as has occurred in other streptococci, could be the first step to achieving high-level fluroquinolone resistance under antibiotic pressure. Other resistance rates could be explained by the transferable resistance mechanisms described above. Aside from the elements carrying tetracycline resistance, most of the isolates also had other resistance mechanisms carried by other elements (Figure 1). Among isolates carrying Tn916, one strain had the erm(T) gene in a non-self-transmissible plasmid and the dfr(F) in a different IME; two isolates had the Tn916 inserted into a larger ICE belonging to the Tn1549 family that also had erm(TR); a single isolate had the dfr(F) in a different IME; one isolate had the macrolide efflux genetic assembly (MEGA) element carrying the mef(E) gene; and finally, one isolate also had erm(B) located near an insertion sequence. On the other hand three out of 12 isolates carrying the IME_SDSE_HUB-4529 also had the erm(TR) gene located in a Tn1549-family of ICE.

Table 1 shows the antibiotic susceptibility of the tetracycline-resistant SDSE. Beta-lac tams (penicillin and cefotaxime), vancomycin, and linezolid were active against all the strains. In fact, beta-lactams are the first-line treatment of SDSE infections, and linezolid could be used as an alternative treatment in case of allergy. Regarding levofloxacin, al strains were "susceptible, increased exposure" following the EUCAST criteria. Neverthe less, one strain had a D83A substitution in ParC and had a MIC of levofloxacin of 2 mg/L which, as has occurred in other streptococci, could be the first step to achieving high-leve fluroquinolone resistance under antibiotic pressure. Other resistance rates could be ex plained by the transferable resistance mechanisms described above. The phylogenetic analysis of the tetracycline-resistant isolates showed a high STs' and emm-type diversity. We found 10 different STs (ST15 n = 11, ST4 n = 2, ST127 n = 2, and ST8, ST27, ST31, ST141, DLV8, SLV15, SLV38 n = 1), and 13 different emmtypes (stC839.2 n = 4, stG652.0 n = 3, stG480.0 n = 2, stG6.1 n = 2, stG10.0 n = 2, stG245.0 n = 2, and stG2574.3, stG166b.0, stG6746.0, stC36.9, emm131.0, stC5345.1, stG97.0 n = 1) among tetracycline-resistant strains harboring the tet(M). These STs and emm-types have been previously described as a cause of disease in humans [START_REF] Lu | Molecular Characterization and Antibiotic Resistance of Clinical Streptococcus dysgalactiae subsp. equisimilis in Beijing, China[END_REF][START_REF] Rojo-Bezares | Streptococcus dysgalactiae subsp. equisimilis from Invasive and Non-Invasive Infections in Spain: Combining Epidemiology, Molecular Characterization, and Genetic Diversity[END_REF][START_REF] Gherardi | Genetic Diversity and Virulence Properties of Streptococcus dysgalactiae subsp. equisimilis from Different Sources[END_REF][START_REF] De Souza | Antimicrobial Susceptibility and Genetic Relationships among Streptococcus dysgalactiae subsp. equisimilis Isolates in Rio de Janeiro[END_REF][START_REF] Leitner | Prevalence of Emm Types and Antimicrobial Susceptibility of Streptococcus dysgalactiae subsp. equisimilis in Austria[END_REF][START_REF] Traverso | Molecular Characterization of Invasive Streptococcus dysgalactiae subsp. equisimilis. Multicenter Study: Argentina 2011-2012[END_REF][START_REF] Loubinoux | Adult Invasive and Noninvasive Infections Due to Streptococcus dysgalactiae subsp. equisimilis in France from 2006 to 2010[END_REF]. Figure 1 shows the phylogenetic relatedness of the SDSE strains carrying tet(M) regardless of the MGEs carrying this gene. Of note, the isolates carrying the tet(M) gene in the ICEs of the Tn916 family were genetically more distinct (Figure 1) than the strains carrying tet(M) in the novel IME. In fact, all isolates carrying the IME_SDSE_HUB-4529 belonged to the same clonal complex (CC15) suggesting a clonal spread of resistance. Nevertheless, these strains of CC15 had different emm-types. In fact, no clear relationship between emm-type and clonal complex has been established for SDSE, as occurs for S. pyogenes, highlighting the more accurate role of CC to differentiate SDSE lineages. Some tetracycline-resistant isolates were also resistant to macrolides, lincosamides, and trimethoprim-sulfamethoxazole. These co-resistances were more frequent in the strains carrying Tn916 (7/10) than in the ones that carry the IME_SDSE_HUB-4529 (3/12) (Figure 1).

MGEs are a frequent cause of resistance dissemination in streptococci. Among them, the ICEs had all the genes required for conjugation and could easily spread intra-and interspecies [START_REF] Ambroset | New Insights into the Classification and Integration Specificity of Streptococcus Integrative Conjugative Elements through Extensive Genome Exploration[END_REF]. In this way, we found a high clonal diversity of tetracycline-resistant strains harboring the tet(M) gene in the Tn916. Furthermore, this element has been associated with resistance in streptococci since the late 1990s, enhancing the opportunities to spread among different strains and species [START_REF] Rice | Tn916 Family Conjugative Transposons and Dissemination of Antimicrobial Resistance Determinants[END_REF]. In contrast, we found a high genetic relatedness among isolates having the tet(M) gene in the new IME (ST15), highlighting that the increase in the prevalence of this new element could be associated preferably to clonal spread of the successful lineages having them. Nevertheless, the presence of ICEs harboring other resistance genes in some of the strains suggests a putative scenario of co-dissemination among other streptococci. Furthermore, it is important to note that other ICEs lacking resistance determinants could also be found among strains and could play a role in the spreading of IMEs [START_REF] Guédon | The Obscure World of Integrative and Mobilizable Elements, Highly Widespread Elements That Pirate Bacterial Conjugative Systems[END_REF].

In conclusion, the spread of tetracycline resistance in Streptococcus dysgalactiae subsp. equisimilis is mediated by both the horizontal spread of mobile genetic elements among different strains and by the clonal spread of tetracycline-resistant lineages harboring tet(M). We described a new integrative mobilizable element in Streptococcus dysgalactiae subsp. equisimilis with a putative origin from E. faecium or C. difficile, which highlights the importance of interspecies transfer of resistance determinants.

Materials and Methods

This was a retrospective study from 2012 to 2019 including all tetracycline-resistant iSDSE in the Hospital Universitari de Bellvitge (HUB), an adult tertiary teaching hospital located in the Southern Barcelona area, Spain. Furthermore, in order to understand the spread of MGEs and tetracycline-resistant lineages around the country, isolates causing iSDSE in three other hospitals during 2018 were also included. These additional hospitals were in the Center (H. Universitario Gregorio Marañón, Madrid, HGUGM), North (H. Universitario Donostia, Donostia, HUD), and East (Consorci Sanitari Parc Taulí, Sabadell, CSPT) of Spain.

The strains were identified by Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF, Bruker-Daltonics, Bremen, Germany) and tested by both discdiffusion and broth microdilution methods for antimicrobial susceptibility following the EUCAST recommendations and criteria (www.eucast.org (accessed on 10 October 2022)). For microdilution, the commercially available plates were used (STRHAE, Sensititre, Ther-moFisher Scientific, Waltham, MA, USA). The following antibiotics were tested: penicillin, ampicillin, cefotaxime, erythromycin, clindamycin, linezolid, tetracycline, chloramphenicol, ciprofloxacin, levofloxacin, vancomycin, and trimethoprim-sulfamethoxazole.

The molecular characterization of tetracycline-resistant strains was performed by whole genome sequencing. Genomic DNA was extracted using a QIAamp DNA Mini Kit (Qiagen) and quantified with the QuantiFluor dsDNA System (Promega Corporation, Madison, WI, USA). The libraries were prepared using Nextera XT and sequenced by paired-end sequencing (2 × 150 bp) on an Illumina MiSeq Platform (Illumina Inc., San Diego, CA, USA), following the manufacturer's instructions. The read quality assessment and genome assembly was conducted using the INNUca v4.2 pipeline (github.com/B-UMMI/INNUca (accessed on 10 October 2021)). The multilocus sequence type (MLST) was in silico deduced using the MLST v2.4 software (github.com/tseemann/mlst (accessed on 10 October 2021)) and the PubMLST database (pubmlst.org/organisms/streptococcus-dysgalactiae (accessed on 0 October 12021)) and the emm-type by the CDC databases (www2.cdc.gov/ vaccines/biotech/strepblast.asp (accessed on 10 October 2021)). The final assemblies were annotated with Prokka v1.13.7, whereas genes related to conjugative or mobilizable genetic elements were annotated with ICEscreen (Lao et al., unpublished results), a tool to detect ICEs and integrative mobilizable elements (IMEs) in Firmicute genomes. The resistance genes were found using the ResFinder database [START_REF] Zankari | Identification of Acquired Antimicrobial Resistance Genes[END_REF]. The genetic environment of the tet(M) gene was studied through comparison with previously described sequences present in public databases using Geneious 9.1.7 (Biomatters, Auckland, New Zealand). For phylogenetic analysis, a single nucleotide polymorphism alignment with Snippy v4.4.0 (github.com/tseemann/snippy (accessed on 11 January 2023)) was performed, the recombination was removed by Gubbins v2.4 [START_REF] Croucher | Rapid Phylogenetic Analysis of Large Samples of Recombinant Bacterial Whole Genome Sequences Using Gubbins[END_REF], and subsequently, a final phylogenetic tree was constructed with RAxML-NG and 100 bootstrap replicates [START_REF] Kozlov | RAxML-NG: A Fast, Scalable and User-friendly Tool for Maximum Likelihood Phylogenetic Inference[END_REF], represented using Microreact (microreact.org/ (accessed on 11 January 2023)). The sequence comparisons between the new IME described in this work and those available on NCBI were displayed using Easyfig program. The sequence data were deposited in the European Nucleotide Archive under the project accession number PRJEB55810 (Supplementary Material Table S1).

Supplementary Materials:

The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antibiotics12030579/s1, Table S1: Antibiotic resistance and mobile genetic elements in the 24 tetracycline-resistant strains. Funding: This study was supported through the Projects PI21/01000, "Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Respiratorias (CB06/06/0037)" and "CIBER de Enfermededes Infecciosas (CB21/13/0009)", an initiative of the Instituto de Salud Carlos III, Madrid, Spain. It was co-funded by the European Regional Development Fund/European Social Fund (ERDF/ESF, "Investing in your future"). We thank the CERCA Programme/Generalitat de Catalunya for institutional support.

Institutional Review Board Statement:

This study was in accordance with the Declaration of Helsinki from the World Medical Association. It was approved by the Clinical Research Ethics Committee of Bellvitge University Hospital (PR283/21). Written informed consent was not required as this was a retrospective and observational study with isolates obtained as part of routine microbiological tests. Patient confidentiality was always protected, and all personal data were anonymized following the current legal normative in Spain (LOPD 15/1999 and RD 1720/2007). Moreover, this project followed Law 14/2007 on Biomedical Research for the management of biological samples in clinical research.

Informed Consent Statement: Patient consent was waived because acces to electronic clinical records of patients was retrospective, the study involved no intervention, and the results would not be accurate without the collection of all episodes.

Figure 1 .

 1 Figure 1. Phylogenetic tree and presence/absence matrix depicting the acquired resistance genes of iSDSE carrying tet(M). The strain names are shown on the tree branches. The colored squares in the first column represent the ST (Sequence-Type). The second column shows the emm-types. The two first columns of light blue and grey squares represent the mobile genetic elements (MGEs) in which the tet(M) gene is found. The following columns represent a presence/absence matrix of tet(M) and other acquired resistance genes indicating in dark blue when present in the same element as tet(M) and in dark red when present in a different element.

Figure 1 .

 1 Figure 1. Phylogenetic tree and presence/absence matrix depicting the acquired resistance genes of iSDSE carrying tet(M). The strain names are shown on the tree branches. The colored squares in the first column represent the ST (Sequence-Type). The second column shows the emm-types. The two first columns of light blue and grey squares represent the mobile genetic elements (MGEs) in which the tet(M) gene is found. The following columns represent a presence/absence matrix of tet(M) and other acquired resistance genes indicating in dark blue when present in the same element as tet(M) and in dark red when present in a different element.

Figure 2 .

 2 Figure 2. Schematic representation of the IME containing tet(M) in the SDSE compared with the se quence from E. faecium and C. difficile. This figure shows the structure of the IME containing the tet(M gene found in twelve strains of the present study (HUB, HUD, HGUGM) compared with the previ ously described isolates in the literature for E. faecium (Acc. num AP019408.1) and C. difficile (Acc. num CP016102.1). The size of this IME is 9352 bp. The grey-shaded areas connect identical regions (100% identity). Each arrow represents a gene. Colors represent different genes. Grey: chromosomal gene (compared in SDSE with reference strain (AC-2713) (Acc num HE858529.1)). Red: integrase; orange excisionase; yellow: MOBT relaxase; green: tet(M) gene; purple: unknown function genes.

Figure 2 .

 2 Figure 2. Schematic representation of the IME containing tet(M) in the SDSE compared with the sequence from E. faecium and C. difficile. This figure shows the structure of the IME containing the tet(M) gene found in twelve strains of the present study (HUB, HUD, HGUGM) compared with the previously described isolates in the literature for E. faecium (Acc. num AP019408.1) and C. difficile (Acc. num CP016102.1). The size of this IME is 9352 bp. The grey-shaded areas connect identical regions (100% identity). Each arrow represents a gene. Colors represent different genes. Grey: chromosomal genes (compared in SDSE with reference strain (AC-2713) (Acc num HE858529.1)). Red: integrase; orange: excisionase; yellow: MOBT relaxase; green: tet(M) gene; purple: unknown function genes.

  Figure S1; Schematic representation of the IME containing tet(M) in SDSE (SDSE-HUB-4529) compared with different sequences from E. faecium, C. difficile, Clostridium innocuum, E. faecalis, and Catenibacterium sp. Author Contributions: Conceptualization, C.A., G.L.d.E. and N.L.-B.; methodology, G.L.d.E., A.G.-D., D.B., G.G. and J.L.; software, A.G.-D., J.L., H.C., T.L. and N.L.-B.; validation: G.L.d.E., A.G.-D. and C.A.; formal analysis, A.G.-D., G.G., J.L., N.L.-B., H.C. and T.L.; investigation, G.L.d.E., A.G.-D., D.B., A.C., E.C., J.M.M. and L.F.-D.; resources, M.Á.D. and C.A.; data curation, G.L.d.E., A.G.-D., G.G. and J.L.; writing-original draft, preparation, G.L.d.E., A.G.-D. and C.A.; writing-review and editing, G.L.d.E., A.G.-D., G.G., J.L., D.B., A.C., J.M.M., E.C., L.F.-D., H.C., T.L., M.Á.D., N.L.-B. and C.A.; visualization, D.B. and G.L.d.E.; supervision, C.A. and N.L.-B.; project administration, C.A.; funding acquisition, C.A. All authors have read and agreed to the published version of the manuscript.

Table 1 .

 1 The antibiotic susceptibility of 24 tetracycline-resistant SDSE strains.

	Antibiotic	% Susceptible	MIC (50) [mg/L]	MIC (90) [mg/L]
	Penicillin	100	<0.03	<0.03
	Cefotaxime	100	<0.06	<0.03
	Erythromycin	54.2	<0.25	>4
	Clindamycin	58.3	<0.25	>0.5

Table 1 .

 1 The antibiotic susceptibility of 24 tetracycline-resistant SDSE strains.

	Antibiotic	% Susceptible	MIC (50) [mg/L]	MIC (90) [mg/L]
	Penicillin	100	<0.03	<0.03
	Cefotaxime	100	<0.06	<0.03
	Erythromycin	54.2	<0.25	>4
	Clindamycin	58.3	<0.25	>0.5
	Linezolid	100	<1	-
	Tetracycline	0	-	>4
	Chloramphenicol	100	2	2
	Levofloxacin	100 *	<1	<1
	Vancomycin	100	<0.5	<0.5
	Trimethoprim-Sulfamethoxazole	91.7	<0.5/9.5	<0.5/9.5

* "Susceptible, increased exposure".

Acknowledgments:

We wish to thank the past and present staff of the Microbiology Departments who contributed to this project on a daily basis. This study was partially presented as a poster at the 21st Lancefield International Symposium for Streptococci and Streptococcal Diseases 2022, Stockholm.

Data Availability Statement: Sequence data were deposited in the European Nucleotide Archive under the project accession number PRJEB55810.

Conflicts of Interest:

The authors declare no conflict of interest.


Y Le Guennec
email: yannis.leguennec@enserg.fr

G Maury

B Cabon

Improvement of dispersion resistance in analog radio-on-fiber upconversion links

Keywords: Optical fiber communications, optical fiber dispersion, analog upconversion, optical microwave mixing, Mach Zehnder modulator, passive unbalanced interferometer

Dispersion fiber sensitivity in optical microwave upconversion links is investigated. Simulations are carried out to calculate output optical spectra and to evaluate mixing power as a function of fiber length for different upconversion systems using Dual Drive/standard Mach Zehnder Modulators or an unbalanced Mach Zehnder interferometer (UMZ). The latter solution prevents complete cancellation of mixing power because of the asymmetric double sideband optical spectrum detected at the output of the UMZ. It is shown that an optimum value of the linewidth enhancement factor of the laser diode can be found to minimize degradations of the mixing power along the fiber.

I. INTRODUCTION

External Mach Zehnder modulators (MZM) are widely used in conventional optical intensity modulation (IM).

For example, the optical carrier can be modulated in the RF frequency range by a local oscillator (LO) signal to generate an optical field that includes the optical carrier and two modulation sidebands (it refers to the so called Double Sideband (DSB) modulation). If the signal is transmitted over the fiber, chromatic dispersion causes each sideband of the continuous wave (CW) modulation signal to have a different phase shift depending on the length of the fiber, dispersion fiber parameter and LO frequency modulation. At the optical receiver, each sideband beats with the optical carrier and the two beat signals ideally interfere constructively. But, when the phase shift reaches  because of dispersion group delays, the two beat signals cancel each other which results in a power degradation of the LO signal. The higher the LO frequency is, the shorter the fiber length for LO power cancellation is. Chromatic dispersion can be almost totally overcome with Single Sideband (SSB) modulation using a Dual Drive Mach-Zehnder Modulator (DD-MZM) biased at quadrature [1]. One of the two optical sidebands of the CW modulated signal is simply suppressed and total cancellation of LO power is no longer possible.

MZM are also often used to generate up and down frequency conversion of microwave (MW) signals, because they avoid insertion loss due to E/O and O/E conversions [START_REF] Smith | Overcoming Chromatic-Dispersion Effects in Fiber-Wireless Systems Incorporating External Modulators[END_REF]. Moreover upconversion allows to generate higher frequency than that which could be obtained by simple direct modulation of a laser diode. Many applications have been found in the fields of defence radar and telecom systems in which intermediate RF frequencies must be generated from an LO signal [START_REF] Seeds | Optical Technologies for Phased Array Antennas[END_REF]. These conventional solutions using MZM for generation of MW mixing are expensive and we have reported yet a solution based on a quite unexpensive UMZ [4]. In this solution, which uses a passive component, a conversion gain can be obtained in addition, which represents a great advantage over traditional methods that use active MZM. As for IM, the generation of up-converted MW signals at high frequency suffer also from periodic degradation of the detected upconverted signal power after propagation along a dispersive fiber.

In this article, the most widely used techniques for optical MW mixing of CW signals are exposed and analyzed in terms of dispersion resistance. The use of MZM or DD-MZM biased at quadrature as upconverters of intermediate frequency (IF) modulation has been yet analyzed theoretically [START_REF] Corral | General Expressions for IM/DD Dispersive Analog Optical Links With External Modulation or Optical Upconversion in a Mach Zehnder Electrooptical Modulator[END_REF]. Dispersion resistance of these solutions is here compared to an MZM operating in a non linear regime when both MW signals (IF and LO) are applied to the modulator. Our original method using the UMZ with direct frequency modulation (FM) of a DFB laser diode (LD) converted into IM is studied for the first time in terms of dispersion resistance. Direct analysis of the output optical spectra of the different MW mixing devices is made to discuss dispersion sensitivity.

Simulations of mixing power as a function of fiber length are carried out for all these optical mixing systems (DD-MZM, MZM and UMZ). It is shown that the common solutions have more sensitivity to fiber chromatic dispersion than the solution based on UMZ. This paper is organized as follows. In section II, the general expressions of optical field spectra at the output of optical mixing systems using MZM, DD-MZM are revised and compared in terms of dispersion tolerance.

Simulation method and results are presented. In section III, the use of UMZ is explained and simulation results are compared to conventional solutions using MZM or DD-MZM. Finally, optimum conditions to use UMZ are discussed and a study of the influence of the linewidth enhancement factor  of the laser diode on the degradation of mixing power is carried out.

II. MZM AND DD-MZM AS UPCONVERTERS

Dispersion sensitivity is simulated here for a fiber dispersion parameter D=17 ps/km/nm at the optical wavelength opt=1.55 m. Calculations are made in the Fourier domain using the expression of the optical transfer function Hfib(f) of the fiber :

  ) 1 ( exp ) ( 2 2           c f f DL j f H opt opt fib  
where f is the optical carrier frequency when the LD is modulated by a LO at frequency fLO, fopt is the optical carrier frequency emitted by the unmodulated LD, L is the length of the fiber, c is the speed of light in vacuum.

The spectrum of the optical field Eout(f) is then obtained by multiplying Hfib(f) with the Fourier Transform of the optical field E(t) at the output of the mixing device [2]:

) 2 ( )) ( ( ) ( ) ( t E FT f H f E fib out  
Finally, after an Inverse Fourier Transform of Eout(f), the mixing power Pmix at the output of the fiber is obtained by analyzing the spectrum Iout(f) after quadratic photodetection :

) 3 ( )) ( ) ( ( ) ( t E t E FT f I out out out

   a) Upconversion with IF intensity modulation on a first MZM + LO modulation applied to a second MZM or DD-MZM (MZM+MZM or MZM+DD-MZM)

A DD-MZM converter is biased at each arm of the interferometer as shown on Fig. 1. General expressions for applied voltages V1(t) and V2(t) are:

) 4 ( ) 2 sin( ) ( ) 2 sin( ) ( 2 2 2 1 1 1            LO LO LO DC LO LO LO DC t f V V t V t f V V t V    
VDCi is a continuous bias voltage and VLO is the amplitude of a CW local oscillator signal at frequency fLO. A phase shift between electrodes of LO1-LO2=/2 is applied to use the DD-MZM as a SSB up-converter.

Standard MZM is simply a particular case of DD-MZM where LO1-LO2= (DSB upconversion) [START_REF] Corral | General Expressions for IM/DD Dispersive Analog Optical Links With External Modulation or Optical Upconversion in a Mach Zehnder Electrooptical Modulator[END_REF].

The first stage of IF modulation is realized with an ideal MZM modulator biased in linear regime. This modulation is assumed to be ideal with a low intensity modulation index mIF<<1. An LO linear modulation is applied to the second stage of the cascaded upconversion device. Upconversion technique using 2 cascaded MZM will be called MZM+MZM whereas upconversion technique using MZM cascaded with DD-MZM will be called MZM+DD-MZM. Optical field at the output of the upconversion device MZM+MZM or MZM+DD-MZM can be written as:

  ) 5 ( 2 exp ) ( exp ) ( exp ) 2 cos( 2 1 2 ) ( 2 1 t f j V t V j V t V j t f m E t E opt IF IF IF in                                      
where Ein is the amplitude of the optical field delivered at the output of the LD, IF=2fIF , V is the switching voltage of the MZM. Conversion of the IF signal frequency into fIFfLO is only possible at quadrature for MZM+MZM or DD-MZM+MZM. That implies |VDC1-VDC2|=V/2 for both configurations. Frequencies of fIF=1.55 GHz and fLO=4.45 GHz and IM indexes are mIF=0.02 et mLO=0.4 were used. With these frequency values, 2.fIF=3.1 GHz can be easily separated from fLO-fIF=2.9 GHz, and the expected up-converted frequency is fmix=6 GHz. Simulated optical field spectra at the output of MZM+MZM and MZM+DD-MZM are plotted respectively on Fig. 2 and Fig. 3. A symmetric optical field around fopt is observed only in the case of MZM+MZM.

Fig. 4 represents upconversion mixing power at 6 GHz for MZM+MZM configuration compared to MZM+DD-MZM. For the MZM+MZM configuration, first cancellation of the mixing power at L=137 km comes from the beatings at the optical receiver of optical field components which frequency difference equals 6 GHz and which are out of phase (Fig. 2).

i) Beating at the optical receiver of fopt (numbered (1) on Fig. 2) with fopt-6 GHz [START_REF] Smith | Overcoming Chromatic-Dispersion Effects in Fiber-Wireless Systems Incorporating External Modulators[END_REF] written fopt (1) / fopt -6 GHz (2) out of phase with fopt (1) / fopt+6 GHz [START_REF] Seeds | Optical Technologies for Phased Array Antennas[END_REF] ii) fopt-4.45 GHz (4) / fopt +1.55 GHz (5) out of phase with fopt-1.55 GHz (6) / fopt+4.45 GHz [START_REF] Heung | Improvement of Linewidth Enhancement Factor in 1.55-m Multiple-Quatum-Well Laser Diodes[END_REF] iii) fopt-8.9 GHz (8) / fopt-2.9 GHz (9) out of phase with fopt+2.9 GHz (10) / fopt+8.9 GHz (11)

The first cancellation does not occur with MZM+DD-MZM configuration because the symmetry that exists in each beating term in i) ii) and iii) has disappeared in the spectrum of E(t) as shown on Fig. 3. Second cancellation occurs at 395 km for MZM+MZM and MZM+DD-MZM configurations and is caused by the cancellation of the three beating terms together, fopt (1) / fopt-6 GHz (2) with fopt-4.45 GHz (4) / fopt+1.55 GHz [START_REF] Corral | General Expressions for IM/DD Dispersive Analog Optical Links With External Modulation or Optical Upconversion in a Mach Zehnder Electrooptical Modulator[END_REF] and with fopt-8.9 GHz (8) / fopt-2.9 GHz (9).

b) Upconversion with both IF and LO signals applied simultaneously to a single MZM in non linear regime (MZM case)

In this configuration expressions of V1(t) and V2(t) are given by :

               ) 2 sin( ) 2 sin( ) ( ) 2 sin( ) 2 sin( ) ( 2 2 2 2 1 1 1 1 IF IF IF LO LO LO DC IF IF IF LO LO LO DC t f V t f V V t V t f V t f V V t V         (6)
The external MZM operates as a standard modulator LO1-LO2= and IF1-IF2=.

The optical field at the output of MZM is expressed by:

  t f j V t V j V t V j E t E opt in      2 exp ) ( exp ) ( exp 2 ) ( 2 1                       (7)
Conversion of the frequency fIF into fIFfLO is only possible when the MZM operates in the non linear regime (MInimum (MITB) or MAximum (MATB) Transmission Bias point) [START_REF] Corral | General Expressions for IM/DD Dispersive Analog Optical Links With External Modulation or Optical Upconversion in a Mach Zehnder Electrooptical Modulator[END_REF]. When |VDC1-VDC2|=0, MZM operates at MATB and when |VDC1-VDC2|=V, MZM operates at MITB. Definitions of MATB and MITB are given in Fig. 5.

The frequency values and IM indexes are the same as previously used. Optical field spectra at the output of MZM modulated by IF and LO signals show that only fIF and fOL harmonics and intermodulation products of even order are present in the spectrum at MATB whereas only fundamental frequencies remain in the spectrum at MITB (Fig. 6.a and 6.b). Simulation results of Fig. 7 show that first cancellation of mixing power at 6 GHz is obtained with longer fiber length for MITB than for MATB because of the wider spectrum in the case of MATB.

As extreme frequencies are further each other in MATB symmetric spectrum, phase shift between beating terms will increase more rapidly with fiber length.

As shown on Fig. 7, resistance to dispersion is improved with MZM when a narrow DSB symmetric optical spectrum is obtained (MITB case). If we now compare Fig. 4 and7, it can be derived that better performance is obtained with an asymmetric output optical spectrum using MZM+DD-MZM technique. First mixing power cancellation occurs at L=395 km for MZM+DD-MZM against L=137 km for MZM+MZM and L=220 km for MZM. Only one cancellation occurs for MZM+DD-MZM technique for a 800 km fiber link. In the next section, we demonstrate further improvements of resistance to dispersion using an UMZ as an upconverter because this device exhibits an asymmetric DSB output optical spectrum.

III.UMZ AS UPCONVERTER

Principle of optical MW mixing with UMZ has been exposed in previous articles for analog up and down conversion [4] and for conversion of MW subcarriers of digital signals [START_REF] Guennec | Conversions of the Microwave Subcarriers of Digital Signals in Optical Links[END_REF]. This principle is recalled briefly hereafter. The laser diode (LD) is directly modulated by two signals IF and LO. In this paper, we consider an ideal LD linearly intensity modulated:

    t f j t f j t f j t f m t f m E t E opt LO LO LO IF IF IF LO LO LO IF IF IF in LD            2 exp . ) 2 sin( ) 2 sin( exp ) 2 cos( ) 2 cos( 1 ( 2 ) (          (8)
where IF and OL are the frequency modulation index for respectively IF and LO signals. The optical field at the output of the LD ELD(t) (9) holds terms of IM and FM. The LD has typical parameter values: an input impedance of 50 , a linewidth enhancement factor (α) of 5, a threshold current of 25 mA, a biasing current of 50 mA and an emitting power of 1.6 mW. The light at the output of the LD is coupled into a glass substrate integrated UMZ as shown on Fig. 8. Optical MW mixing is obtained converting FM into IM through the UMZ. The optical transfer function of the UMZ HUMZ(f) depends on the free spectral range (FSR) of the interferometer:

) 9 ( ) ] 2 [ exp 1 ( ) ( FSR f j f H UMZ   
The interference regime is fixed by the choice of the optical frequency fopt depending on the bias of the LD.

The spectrum of the optical field at the output of the UMZ E(f) is then expressed by :

) 10 ( )) ( ( ) ( ) ( t E FT f H f E LD UMZ  
Optimum optical MW mixing is obtained working at MATB (=0) or MITB (=) as for MZM but with frequencies respecting condition given by :

    ) 11 ( ' , 2 1 ' 2 , 2 1 2              k FSR k f k FSR k f LO IF
This condition is linked to the fact that the UMZ acts as a linear filter [START_REF] Corral | General Expressions for IM/DD Dispersive Analog Optical Links With External Modulation or Optical Upconversion in a Mach Zehnder Electrooptical Modulator[END_REF]. As FSR is equal to 3 GHz, the input signal frequencies fIF=1.55 GHz and fLO=4.45 GHz are really close to the ideal values 1.5 GHz and 4.5 GHz, these values avoid confusion between intermodulation products. IM indexes are mIF=0.02 and mLO=0.4 as previously. compare dispersion effect with the UMZ and with the MZM (Fig. 7). It shows that minima of mixing power are at exactly the same fiber lengths as for MZM but no deep cancellations occur in the case of UMZ. This great advantage comes from the fact that the UMZ optical field spectra (Fig. 9 and 10) are really similar to MZM spectra (Fig. 6) but with asymmetric amplitudes of spectral lines due to the combined FM and IM of the light at the output of the LD. DSB asymmetric spectra make impossible complete cancellation of the sum of all the beating terms. As for the MZM case, first minimum at MITB occurs after a longer fiber length than at MATB but the minimum is deeper in the MITB case. Then, MATB with the UMZ can be considered as the more resistant case. This better resistance to dispersion is obtained because more asymmetrical spectral lines beat to generate the 6 GHz upconverted signal. Now let us consider the most resistant case only: MATB. Fig. 12 shows simulations of the influence of the linewidth enhancement factor  on maximum fluctuations of the mixing power (Pmax-Pmin) defined on Fig. 11 for different values of input frequencies (fIF=1.55 GHz, fLO=4.45 GHz), (fIF=0.8 GHz, fLO=5.2 GHz) and (fIF=0.2 GHz, fLO=5.8 GHz). All these frequency values lead to the same up-converted frequency of 6 GHz. The linewidth enhancement factor of DFB laser diodes is typically in the range 0.5 to 10 [START_REF] Heung | Improvement of Linewidth Enhancement Factor in 1.55-m Multiple-Quatum-Well Laser Diodes[END_REF]. Fig. 12 shows minimal value for (Pmax-Pmin) for a optimum value of the linewidth enhancement factor α of about 2.7 in the case of quasi ideal frequency values (fIF=1.55 GHz, fLO=4.45 GHz). In this condition, it was demonstrated [START_REF] Guennec | BER Performance Comparison Between Active Mach-Zehnder Modulator and Passive Mach-Zehnder Interferometer for Conversion of Microwave Sub-Carriers of BPSK Signals[END_REF] that we have a mixing gain of about 10log(α 2 )=8.6 dB whereas, in the same conditions of operation, the MZM and DD-MZM solutions would exhibit conversion losses. We notice here that another great interest is that there are very low fluctuations (2.5 dB) of mixing power along the fiber. These results are major advantages compared to classical techniques discussed in section II. When frequencies do not respect (11) fluctuations of Pmix increase dramatically as fast as the frequency values differ from ideal values. For example when fIF=0.2 GHz and fLO=5.8 GHz fluctuations of Pmix reach more than 18 dB.

IV. CONCLUSION

We have compared 3 optical MW upconversion techniques using MZM, DD-MZM or UMZ and we have shown the impact of dispersion on the mixing power. SSB upconversion technique with DD-MZM and first stage intensity modulation leads to asymmetric optical field spectrum. This effect causes a longer fiber length for first mixing power cancellation for the MZM+DD-MZM technique than for MZM+MZM technique. But better results are obtained with the technique using an UMZ and direct FM of an LD as the DSB asymmetrical spectrum at UMZ output prevents total cancellation of mixing power. Deeper analysis has shown that the very small fluctuation of mixing power (2.5 dB) can be obtained for an optimum linewidth enhancement factor of 2.7, with a mixing gain still higher than for the MZM or DD-MZM techniques. A good choice of input frequencies depending on the FSR of the UMZ allows high mixing power and good dispersion tolerance. Sensitive degradation occurs when frequencies do not respect this condition any longer.

Fig 9 .

 9 Fig 9.b and 10.b show simulated optical field spectra at the output of UMZ in the case of MATB and MITB that are in good agreement with measurements (Fig 9.a and Fig. 10.a respectively). Simulation results on Fig. 11

EinFig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 7 Fig

 12347 PhotodetectorDispersive fiber

LIST OF FIGURES


Z Dekali

L Baghli
email: lotfi.baghli@univ-lorraine.fr

Ieee Sm

A Boumediene

Speed controller efficiency of the TSR based MPPT of a variable speed wind power system

Keywords: maximum power point tracking, tip speed ratio, PI controller, sliding mode control, supertwisting

This paper compares the usefulness of linear and nonlinear control laws applied in MPPT based on the TSR algorithm in the wind power system. The main idea of this paper is to show the improvement brought by the higher order sliding mode control based on Supertwisting compared to the conventional PI to mitigate the chattering phenomenon, cancellation of the hyperlink between the controller parameters and the mathematical model of the system and the reduction of the sensitivity to external disturbances. The results obtained confirm the effectiveness of the proposed control laws during all the operating modes of the variable speed wind power system in extracting the available maximum aerodynamic power.

I. INTRODUCTION

Variable speed wind systems can adjust the speed of rotation during changes in wind speed using a partial scale power electronic converter. This concept is based on a double fed induction generator (DFIG) configuration (Figure 1). In this topology, the DFIG rotor is connected to the power grid through partial rate power converters called machine side converter (MSC) and grid side converter (GSC), while the stator of the DFIG is directly connected to the grid through a transformer. The MSC has the role of controlling the rotor currents, torque and rotor speed of the DFIG, while the GSC controls the power flow between the grid and the DFIG rotor [START_REF] Aboudrar | Modeling and Robust Control of a Grid Connected Direct Driven PMSG Wind Turbine By ADRC[END_REF], [START_REF] Blaabjerg | Advanced Control of Doubly Fed Induction Generator for Wind Power Systems[END_REF].

In order to improve the control performance of the wind energy conversion system, we will investigate two control strategies. We take into account the nonlinearities of the model. The first method uses the conventional PI controller, the second is based on a higher order sliding mode control [START_REF] Dekali | Improved Super Twisting Based High Order Direct Power Sliding Mode Control of a Connected DFIG Variable Speed Wind Turbine[END_REF].

The first order sliding mode control has many advantages, such as high robustness and simple design, but faces some drawbacks such as the chattering phenomenon due to the existence of a discontinuous term caused by the sign function in the control input, which contributes to additional heating of the electrical elements and windings and leads to high mechanical stress, thus limiting the practical implementation [START_REF] Gui | Sliding Mode Control With Grid Voltage Modulated DPC for Voltage Source Inverters Under Distorted Grid Voltage[END_REF]. Several literatures has proven that higher-order sliding mode can reduce chattering problems with great efficiency without having limited control accuracy performance and finite-time convergence properties. [START_REF] Guo | Improved super-twisting sliding mode control of a stand-alone DFIG-DC system with harmonic current suppression[END_REF].

The second-order sliding mode control includes the Supertwisting algorithm among its classes, which is applicable to systems that have a relative degree of 1, this technique acts on the higher order time derivatives of the sliding manifold, [START_REF] Rached | DSP in the Loop Implementation of Sliding Mode and Super Twisting Sliding Mode Controllers Combined with an Extended Kalman Observer for Wind Energy System Involving a DFIG[END_REF].

The focus of this work is to improve the efficiency of the MPPT by improving the performance of the speed control loop, and consequently, to increase the production of the generated power.

The sections of this work are presented as: Section II describes the static-dynamic wind turbine model. After, the proposed control techniques were investigated in section three. The results obtained were analyzed and interpreted in the following section. The paper ends with conclusions.

II. THE STATIC-DYNAMIC WIND TURBINE MODEL

The simplified wind turbine model can be generalized to two main sub-models, a static model that defines the available power of the wind system and a dynamic model that describes the mechanical behavior of the aerodynamic system. Along with the diameter and altitude of the wind blades, the power coefficient C p are major factors in characterizing the energy productivity of the system. This parameter is often given by manufacturers and gives the ratio between the angle of inclination of the blade β and the relative speed of the turbine λ [START_REF] Dekali | Experimental Emulation of a Small Wind Turbine Under Operating Modes Using DC Motor[END_REF].

(1) With:

(2)

Where R is the blade radius, V the wind speed, and Ω t is the mechanical speed of the system.

The dynamic model shows the opposing forces applied by the aerodynamic torque T t and the torque of the generator T em on the turbine shaft passing through the gearbox. The dynamic model with one mass that brought back on the slow shaft is made up of single inertia and a single friction coefficient gathering all the external friction coefficients. The fundamental mathematical relation of this model is given by [START_REF] Martinez | Open loop wind turbine emulator[END_REF]:

(3) Fig. 1. Control structure of the DFIG -variable speed wind power system Where J t is the moment of inertia of the turbine and f is the friction coefficient of the system and G is the gear ratio. Figure 2 shows the distribution of forces applied to the aerodynamic drive shaft. Considering the rotor speed and power output limits of a wind system, the variable speed wind turbine operating zones includes three important zones [START_REF] Blaabjerg | Advanced Control of Doubly Fed Induction Generator for Wind Power Systems[END_REF]. The Tip Speed Ratio (TSR) based Maximum Power Point Tracking (MPPT) is used in wind power systems to maintain the ratio λ at its an optimal value, and thus obtain the maximum power coefficient in order to extract the maximum dynamic power available on the drive shaft [START_REF] Chen | Design of robust MPPT controller for grid-connected PMSG-Based wind turbine via perturbation observation based nonlinear adaptive control[END_REF]. The principle of this technique is the adjustment of the rotor speed according to the variation of the wind speed. The rotor speed varies proportionally to the wind speed. The wind turbine is controlled at the optimal turbine speed generated by the controller based on the current wind speed [START_REF] Dekali | Improved hardware implementation of a TSR based MPPT algorithm for a low cost connected wind turbine emulator under unbalanced wind speeds[END_REF].

A. Applied TSR based MPPT with PI structure

The PI controller is used to reduce the error between the optimal speed reference and the applied speed. The narrow operating mode zones present the big drawback of this algorithm, as well as the additional cost of the anemometer which is very important to provide the best wind speed measurement [START_REF] Dekali | Real-Time Emulation of a Grid-Connected Wind Energy Conversion System Based Double Fed Induction Generator Configuration under Random Operating Modes[END_REF]. The PI speed controller output impose the electromagnetic torque reference T em * . The PI controller ensures the convergence of the speed to its reference. The parameters of the PI controller are calculated according to the mechanical characteristics of the generator. Parametric variations of these parameters can limit controller performance

B. Applied TSR based MPPT with SMCST structure

The Super Twisting algorithm is a second-order class of the sliding mode, which has the possibility of reducing chattering problems compared to the sliding mode of the first order, it can be applied directly when the system is of a relative degree of the first, it is applicable to the system which has a relative degree of 1 [START_REF] Yaichi | Super-twisting Sliding Mode Control of a Doubly-fed Induction Generator Based on the SVM Strategy[END_REF]. The algebraic formula for the mechanical speed control law is given by:

(5)

The general diagram of the SMC Supertwisting speed control technique is simplified in the following figure.

IV. RESULTS AND DISCUSSIONS

In this part, we compare, by simulation, the reliability of the proposed control laws for applying the MPPT algorithm based on the TSR principle. The applied wind speed profile (Fig. 7) provides the slow transmutation between the operating modes, the latter gives good behavior for the control loop robustness test.

All the simulation tests were carried out on a low power wind system, table II gives its nominal parameters. Better tracking accuracy is achieved by both controllers (Fig. 8), except for a slight disparity when increasing or decreasing the reference. The tracking error of the speed control loop is minimized thanks to the performances of the controllers (Fig. 9); thus, they are able to control correctly. However, fluctuations and high loads can lead to an imbalance of the device which require a mechanical system to absorb these noises. The instantaneous changes in the wind speeds generate a sudden variation in the reference torque T em * to regulate the speed. The quality of the power and the torque (Fig. 10 and11) is affected. In the case of SMCST controller, this phenomenon is amplified, because of the control law discontinuity. This gives more advantage to the linear controller in this kind of systems. The performance of the system control can be quantitatively evaluated using performance indices, such as the Integral Absolute Error (IAE) and the Integral Square Error (ISE). These indices are used for the purpose of quantifying the dynamics of the error between the setpoint and the system output, where:

(5)

The following table I shows the EAI and ESI values obtained from simulation results. These indices indicate the stability of the system as a function of the accumulation of controller output errors, the closer these values are to zero, the better the control. In general, the statistical comparison gives a proximity between the indices of the two controllers, with a slight advantage of the linear PI controller.

CONCLUSION

In this paper, a comparative study is conducted to realize the MPPT based on the TSR technique. Linear and non-linear control laws are applied to the speed control loop to manage the extraction of the maximum power.

The effectiveness and applicability of the proposed controllers have been evaluated by simulation tests. The main advantages of the higher order sliding mode control are the good tracking accuracy, the ease of the numerical implementation of the control algorithm, as well as the total independence of the controller gains to the parameter variations of the system. Nevertheless, chattering remains the major disadvantage of this technique which may present physical limitations for real time experimental realization.

The comparison between the results of PI control and those of supertwisting one shows a great resemblance based on the dynamic behavior. However, even if the sliding mode regulation has the advantage of robustness and ease of implementation, the PI controller is distinguished by lower error indices, which explains the wide use of this type of controller in wind power systems and also in other industrial applications. The advantages shown by this paper confirm that conventional PI controllers will be largely sufficient for this type of system.

APPENDIX TABLE II. WIND TURBINE SIETINGS

Setting Parameter

Air density 1.225

Gearbox ratio 7

Blade radius 3

Nominal speed 1500

Nominal power 1.5

Fig. 2 .

 2 Fig. 2. Dynamic model with one mass brought back on the slow shaft

Fig. 3 .

 3 Fig. 3. Operating zones of a variable speed wind turbine

Fig. 4 .

 4 Fig. 4. TSR based MPPT principle, (a) TSR based MPPT principle (b) TSR based MPPT operating zone

Fig. 5 .

 5 Fig. 5. MPPT basd TSR using PI controller

Fig. 6 .

 6 Fig. 6. MPPT basd TSR using SMCST controller

Fig. 7 .

 7 Fig. 7. Applied wind speed profile

Fig. 8 .

 8 Fig. 8. Tracking accuracy of the speed control loop

Fig. 9 .

 9 Fig. 9. Tracking error of the speed control loop

Fig. 10 .Fig. 11 .

 1011 Fig. 10. Resultant active power reference

TABLE I

 I

		.	CONTROLLERS INDICES
			PI	SMCST
	IAE	13.57	22.74
	ISE	575	670