Xin Tan

Elodie Ramond

Anne Jamet

Jean-Philippe Barnier

Baptiste Decaux-Tramoni

Marion Dupuis

Daniel Euphrasie

Fabiola Tros

Ivan Nemazanyy

Jason Ziveri

Xavier Nassif

Alain Charbit
email: alain.charbit@inserm.fr

Mathieu Coureuil
email: mathieu.coureuil@inserm.fr

Transketolase of Staphylococcus aureus in the Control of Master Regulators of Stress Response During Infection

Keywords: Transketolase, Staphylococcus aureus, metabolic adaptation, Pentose Phosphate Pathway, sigma B, RpiRc

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Background

Staphylococcus aureus is a leading cause of severe skin and soft tissue infections, pneumonia, endocarditis and bone and joint infections [1,2]. S. aureus invades and survives within eukaryotic host cells [3][4][5][6][7][8][9]. This is likely to play an important role in chronicity and treatment failures [10][START_REF] Kahl | Population dynamics of persistent Staphylococcus aureus isolated from the airways of cystic fibrosis patients during a 6-year prospective study[END_REF][START_REF] Fisher | Persistent bacterial infections and persister cells[END_REF]. To evade host defences and silently persist within human cells [START_REF] Grosz | Cytoplasmic replication of Staphylococcus aureus upon phagosomal escape triggered by phenol-soluble modulin alpha[END_REF][START_REF] Korea | Staphylococcal Esx proteins modulate apoptosis and release of intracellular Staphylococcus aureus during infection in epithelial cells[END_REF], S. aureus can dynamically switch phenotypes from a highly aggressive and cytotoxic phenotype to a metabolically inactive phenotype associated with decrease TCA activity [START_REF] Kohler | A defect in menadione biosynthesis induces global changes in gene expression in Staphylococcus aureus[END_REF][START_REF] Kriegeskorte | Small colony variants of Staphylococcus aureus reveal distinct protein profiles[END_REF][START_REF] Kriegeskorte | Staphylococcus aureus small colony variants show common metabolic features in central metabolism irrespective of the underlying auxotrophism[END_REF][START_REF] Tuchscherr | Staphylococcus aureus dynamically adapts global regulators and virulence factor expression in the course from acute to chronic infection[END_REF]. Reprogramming of the TCA cycle notably involves the glycolysis/gluconeogenesis and pentose phosphate pathway (PPP) [START_REF] Stincone | The return of metabolism: biochemistry and physiology of the pentose phosphate pathway[END_REF].

The contribution of glycolysis/gluconeogenesis to intracellular persistence of S. aureus has been recently studied [START_REF] Harper | Staphylococcus aureus Responds to the Central Metabolite Pyruvate To Regulate Virulence[END_REF][START_REF] Purves | Comparison of the regulation, metabolic functions, and roles in virulence of the glyceraldehyde-3-phosphate dehydrogenase homologues gapA and gapB in Staphylococcus aureus[END_REF][START_REF] Vitko | Glycolytic dependency of high-level nitric oxide resistance and virulence in Staphylococcus aureus[END_REF][START_REF] Vitko | Expanded Glucose Import Capability Affords Staphylococcus aureus Optimized Glycolytic Flux during Infection[END_REF]. In contrast, the precise role of the PPP is unexplored. The PPP is important for producing nucleotide precursors for DNA synthesis and repair as well as precursors for aromatic amino acids or peptidoglycan synthesis. In addition, the oxidative branch of the PPP contributes to bacterial tolerance to oxidative stress by generating the redox molecule NADPH/H + , which is the substrate for other reducing agents such as glutathione. The activity of the PPP has been shown to be frequently increased in Gram-positive pathogens in response to environmental stresses and in infection models [START_REF] Gardner | Metabolic Mitigation of Staphylococcus aureus Vancomycin Intermediate-Level Susceptibility[END_REF][START_REF] Fleury | Transcriptomic and metabolic responses of Staphylococcus aureus exposed to supra-physiological temperatures[END_REF][START_REF] Joseph | Comparative genome biology of a serogroup B carriage and disease strain supports a polygenic nature of meningococcal virulence[END_REF] or in some hospitalacquired isolates [START_REF] Mekonnen | Metabolic niche adaptation of community-and hospital-associated methicillin-resistant Staphylococcus aureus[END_REF]. While it has been suggested that the transcriptional regulator RpiRc might control the PPP [START_REF] Zhu | RpiR homologues may link Staphylococcus aureus RNAIII synthesis and pentose phosphate pathway regulation[END_REF][START_REF] Gaupp | RpiRc Is a Pleiotropic Effector of Virulence Determinant Synthesis and Attenuates Pathogenicity in Staphylococcus aureus[END_REF][START_REF] Balasubramanian | Staphylococcus aureus Coordinates Leukocidin Expression and Pathogenesis by Sensing Metabolic Fluxes via RpiRc[END_REF], no gene regulation has been yet associated with any change in its activity in S. aureus.

Here, we study the role of PPP during the intracellular persistence of S. aureus by focusing on one of the key enzyme of this pathway, transketolase (TKT). tkt genes are found in a wide range of organisms including bacteria, plants and mammals, underlining the ubiquitous role of this enzyme in biology. We show here that tkt inactivation in S. aureus USA300 leads to deregulation of whole cell metabolism. This correlates with impaired proliferation of intracellular staphylococci in vitro and kidney colonisation defect in infected mice. Unexpectedly, we found that the tkt defective mutant was unable to regulate the two master regulators of virulence RpirC and SigB, leading to a dramatic increase of RNAIII during intracellular invasion of human cells. Altogether our data suggest a major role of the PPP in the control of S. aureus virulence.

Methods

Strains, Cells, Culture conditions and Infection. S. aureus USA300-LAC (designated USA300-WT) was provided by the Biodefense and Emerging Infections Research Resources (BEI). The GFP-expressing strain, the tkt mutant strain and the complemented strain were constructed and cultured as described in supplementary methods. Strains and plasmids used are summarized in supplementary methods.

S. aureus were grown in brain heart infusion (BHI) or chemically defined medium (CDM) supplemented with ribose or fructose or glycerol when it was required [START_REF] Hussain | A chemically defined medium for slime production by coagulase-negative staphylococci[END_REF].

EA.hy926 endothelial cells (ATCC CRL-2922) were grown in DMEM 10 % FBS (5 % CO 2 at 37 °C). When indicated, cells were incubated with no glucose DMEM supplemented with glucose, ribose or fructose at a concentration of 25mM. EA.hy926 expressing nuclear mKate2 were transduced with the IncuCyte ® NucLight Red Lentivirus.

Cells were infected at a multiplicity of infection (MOI) of 1 for 1 h and washed three times with 100 µL of phosphate buffer saline (PBS) containing 300 µg/ml gentamicin to remove extracellular bacteria. Cells were then incubated in PBS-gentamicin (50µg/ml). Gentamicin is an antibiotic with a high bactericidal effect on S. aureus USA300 (CMI=2 µg/ml) and a very poor penetration inside eukaryotic cells. Its use was necessary to abolish extracellular proliferation.

LAMP-1 colocalization assay in EA.hy926 infected cells is described in the supplementary methods.

Time lapse microscopy

EA.hy926 cells expressing nuclear mKate2 were seeded in ImageLock 96-well plates (Essen BioScience Inc). Cells were infected with WT and tkt mutant of the GFP-expressing USA300 strain. Plates were incubated and monitored at 5% CO2 and 37°C for 24 hours in the fully automated microscope Incucyte ® S3 (Essen BioScience). The detailed analysis is further described in the supplementary methods.

Transcriptional analyses

The bacteria were recovered from lysed cells or planktonic overnight culture. Nucleic acids were released by resuspending bacteria in TE buffer containing lysostaphin. RNAs recovery, reverse transcription and PCR were performed as described in the supplementary methods.

We used the "The Relative Standard Curve Method" for analyzing the qRT-PCR data.

Metabolomic analyses

Metabolite profiling of S. aureus isolates grown to stationary phase in BHI Broth was performed by liquid chromatography-mass spectrometry (LC-MS) as described in the supplementary methods [9,[START_REF] Mackay | Analysis of Cell Metabolism Using LC-MS and Isotope Tracers[END_REF][START_REF] Chong | MetaboAnalyst 4.0: towards more transparent and integrative metabolomics analysis[END_REF].

Transketolase activity assay

TKT activity was analysed on bacteria grown to stationary phase in BHI as described in the supplementary methods [START_REF] Shaw | Salmonella enterica serovar Typhimurium has three transketolase enzymes contributing to the pentose phosphate pathway[END_REF]. TKT activity was expressed as units per microgram of total protein. One unit of enzyme was defined as the amount of enzyme that oxidized 1 µmol of NADH per minute.

In vivo infection

Mice were infected intravenously (iv) in the tail vein as described in the supplementary methods. For bacterial burden determination, mice were euthanized after 4, 24, 48 and 72 hours. Sequential dilutions of homogenized preparations from spleen and kidney were spread onto BHI agar plates.

Statistics

Statistical significances were assessed using one-way analysis of variance (ANOVA) with Dunnett's correction, unpaired two-tail Student's t-test, or Kruskal Wallis test. P values of p<0.05 were considered to indicate statistical significance.

Transketolase inactivation in S. aureus USA300

S. aureus genomes possess a unique transketolase-encoding gene, designated tkt, which is highly conserved within species. Transketolase (TKT) is an enzyme of the nonoxidative branch of the PPP involved in two main reversible enzymatic reactions: i) Fructose-6-P + Glyceraldehyde-3-P <-> Erythrose-4-P + Xylulose-5-P; and ii) Sedoheptulose-7-P + Glyceraldehyde-3-P <-> Ribose-5-P + Xylulose-5-P (Supplementary Figure S2A). Several TKT structures have been solved [START_REF] Fullam | Structure and function of the transketolase from Mycobacterium tuberculosis and comparison with the human enzyme[END_REF] and allow the modelisation of the S. aureus TKT (Supplementary Figure S1B). The protein of S. aureus is 662 amino acid long and harbours three domains (Supplementary Figure S1A). Of note, although ubiquitously expressed in Eukaryotes and Bacteria, S. aureus and Homo sapiens TKT proteins share only 22.4% amino acid identity (Supplementary Figure S1C). We constructed a chromosomal deletion of the tkt gene in S. aureus strain USA300 (∆tkt strain), and generated a complemented strain (designated Cp-tkt) by expressing the wild-type tkt allele preceded by its own promoter in the ∆tkt strain. Gene inactivation was confirmed by quantification of the transketolase activity (Supplementary Figure S2).

The ∆tkt mutant showed only a slight growth decrease compared to the WT strain in complete BHI Broth or in Dulbecco's Modified Eagle Medium (DMEM) (Figure 1A). In contrast, the ∆tkt mutant strain was unable to grow in chemically defined medium [START_REF] Hussain | A chemically defined medium for slime production by coagulase-negative staphylococci[END_REF] supplemented with ribose (which can be converted to Ribose-5-phospate by ribokinase) (Supplementary Figure S2C). In all assays, functional complementation was observed in the Cp-tkt strain.

We next performed a whole cell metabolomic analysis on WT and ∆tkt strains grown for 24h in BHI Broth (i.e., when identical numbers of cells were recorded) to evaluate the global impact of tkt deletion on bacterial metabolism (Figure 1B and Supplementary Figure S3 and Table S1). Remarkably, we observed a massive accumulation of ribose-5-phosphate (R5P; 56-fold increase) concomitant with a decrease in ribose content (4-fold decrease) in the ∆tkt mutant compared to WT. This accumulation was associated with an increased amount of metabolites derived from Ribose-5P, such as inosine-5-monophosphate (IMP), xanthosine-5monophosphate (XMP) and hypoxanthine. We also observed a 2.5-fold decrease in D-sedo-heptulose-7P amount in the ∆tkt mutant compared to WT accompanied by a significant decrease in tyrosine, histidine and tryptophan amounts (5, 10 and 12 fold respectively), three aromatic amino-acids produced from the PPP. The glutamate pathway seemed to be significantly more active in the ∆tkt strain compared to WT as the amounts of glutamate, proline and ornithine were all increased. The higher amounts of glutamate, possibly fuelling the TCA cycle at the level of α-ketoglutarate, could be responsible for the increased expression of some TCA cycle metabolites (i.e., fumarate, malate, succinate and α-ketoglutarate) in the ∆tkt strain.

Of note, ATP content was not altered in ∆tkt strain. These results demonstrate that deregulation of the PPP by tkt inactivation has direct effects on the whole cell metabolism, including aromatic amino acid production, TCA activity and pathways involved in adenine, guanine or peptidoglycan synthesis.

Tkt is essential for intracellular proliferation of S. aureus

Intracellular staphylococci exist as two different populations: bacteria that actively proliferate during the first 24 hrs after invasion, leading to their release in extracellular milieu as a consequence of host cell death, and bacteria that do not replicate. These latter staphylococci can persist within host-cell cytoplasm for several days [3][4][5][6][7][8][9][START_REF] Rollin | Intracellular Survival of Staphylococcus aureus in Endothelial Cells: A Matter of Growth or Persistence[END_REF]. Here we addressed the role of TKT in these two phenotypes in EA.hy926 human endothelial cells by using: i) time lapse-microscopy, to evaluate early intracellular proliferation of S. aureus and ii) colony forming units counting, to monitor the survival kinetics of internalized bacteria over a ten days period.

Time-lapse video microscopy. EA.hy926 cells expressing mKate2 nuclear fluorescent protein were infected by GFP-expressing bacteria. Gentamicin was used throughout the experiment to eliminate extracellular bacteria and prevent re-infection (see Methods), allowing us to solely focus on the fate of intracellular persistent bacteria. Cell infection was monitored using an IncuCyte ® S3 imaging system and images were captured every 30 minutes. Bacterial entry into endothelial cells was similar for WT and ∆tkt strains, showing 0.3~0. with an average number of GFP expressing particles rising from 0.5 to 2 per cells. At the single cell level, WT S. aureus showed active intracellular multiplication, ultimately leading to cell death and bacterial release in the extracellular medium (Figure 2A-B and Movie 1). Thanks to the permanent presence of gentamicin in the medium bacterial release did not lead to proliferation in the extracellular medium. In contrast, the ∆tkt mutant strain showed limited proliferation during the first 24 hours (Figure 2A-B and Movie 2). Finally, using LAMP-1 colocalization assay (Lysosome-associated membrane protein 1), we confirmed that the intracellular ∆tkt strain growth defect was not due to a decreased phagosomal escape (Supplementary Figure S4).

Long-term intracellular survival. Using the same infection conditions as for time-lapse experiment, we monitored survival of internalised Δtkt mutant compared to that of the WT strain at three, seven or ten days after infection by CFU counting. The Δtkt strain behaved likes the WT strain throughout the infection period, ultimately leading to its complete elimination after 10 days (Figure 2C).

Thus, the role of TKT during S. aureus host cell infection seems to be essential for bacteria proliferation and restricted to early time points.

Tkt inactivation is associated with an altered rpiRc and sigB expression.

S. aureus is known to sense its environment and to regulate virulence factors depending on the availability of carbon sources. For example, recent studies have shown that the transcriptional regulator RpiRc could sense metabolic shifts, especially in the PPP, and control expression of RNAIII [START_REF] Gaupp | RpiRc Is a Pleiotropic Effector of Virulence Determinant Synthesis and Attenuates Pathogenicity in Staphylococcus aureus[END_REF][START_REF] Balasubramanian | Staphylococcus aureus Coordinates Leukocidin Expression and Pathogenesis by Sensing Metabolic Fluxes via RpiRc[END_REF]. RNAIII is a key effector in the quorum-sensing system that regulates the expression of a large number of virulence factors [START_REF] Boisset | Staphylococcus aureus RNAIII coordinately represses the synthesis of virulence factors and the transcription regulator Rot by an antisense mechanism[END_REF]. RpiRc is also linked to the alternative Sigma B factor (SigB) that has pleiotropic roles in gene regulation and is a master regulator of intracellular survival [START_REF] Gaupp | RpiRc Is a Pleiotropic Effector of Virulence Determinant Synthesis and Attenuates Pathogenicity in Staphylococcus aureus[END_REF][START_REF] Mader | Staphylococcus aureus Transcriptome Architecture: From Laboratory to Infection-Mimicking Conditions[END_REF][START_REF] Tuchscherr | Sigma Factor SigB Is Crucial to Mediate Staphylococcus aureus Adaptation during Chronic Infections[END_REF].

These data prompted us to follow the expression profile of the master regulator SigB and RpiRc in the WT and the tkt mutant strains during endothelial cells invasion (Figure 3).

EA.hy296 cells were infected as described above to solely focus on intracellular bacteria.

Between day 1 and day 7, sigB and rpiRc transcription increased in the WT strain (by 4-fold and 3-fold, respectively) (Figure 3). In sharp contrast, as early as one day after infection, both genes were significantly down-regulated in the ∆tkt mutant strain compared to WT (8-fold and 7-fold decrease for sigB and rpirC expression, respectively). Consistent with reports demonstrating inhibition of RNAIII expression by RpirC and SigB [START_REF] Tuchscherr | Staphylococcus aureus dynamically adapts global regulators and virulence factor expression in the course from acute to chronic infection[END_REF], we observed a dramatic increase in the expression of RNAIII in the ∆tkt mutant strain (Figure 3), suggesting that the tkt mutant strain is unable to control rpiRc and sigB expression during intracellular survival.

Functional complementation always restored the wild type phenotype (Supplementary Figure S5). To further confirm these data, we monitored the expression of two major regulators of SigB activity, RsbU and RsbW [START_REF] Pane-Farre | Role of RsbU in controlling SigB activity in Staphylococcus aureus following alkaline stress[END_REF]. RsbW, which is co-expressed with sigB, is known to sequestrate and inhibit SigB. RsbU, whose expression is under the control of SigA, is a phosphatase involved in the dephosphorylation of RsbV. In turn, dephosphorylated RsbV (dRsbV) is able to sequestrate RsbW, allowing the release of active SigB. RsbU is thus considered as an activator of SigB, allowing a fine-tuning of SigB activity.

We found that the transcription of both rsbW and rsbU genes was down-regulated in the tkt mutant during infection of endothelial cells. The down-regulation of rsbW is consistent with the down-regulation of its co-expressed gene sigB (Figure 3). The concomitant downregulation of rsbU suggests a global inhibition of the sigB pathway in the tkt mutant strain.

Finally, we followed expression of asp23, a sigB-dependent locus [START_REF] Gertz | Regulation of sigmaBdependent transcription of sigB and asp23 in two different Staphylococcus aureus strains[END_REF]. The recorded downregulation of asp23 transcription in the tkt mutant strain confirmed the inhibition of SigBdependent transcription. All together these transcriptional analyses suggest that the metabolic deregulation observed in tkt strain is sufficient to promote a dramatic reprogramming of the RpiRc and SigB pathways when bacteria invade human cells.

Tkt inactivation impairs proliferation of S. aureus in the kidney of infected mice.

The ability of internalized S. aureus to readily proliferate inside host cell cytoplasm may be seen as an effective mechanism to avoid host innate immunity, ultimately allowing an increased colonization of the infected site. In this respect, it has been previously shown in the mouse model that after intravenous (IV) injection, although rapidly cleared from the bloodstream, bacteria were able to proliferate in the kidney, leading to abscesses formation within the first days of infection [START_REF] Cheng | Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues[END_REF].

Here, we monitored the kinetics of in vivo proliferation of WT, tkt and Cp-tkt strains in kidneys and spleens of BalB/c mice (Figure 4) after IV infection with 10 7 bacteria per mouse.

As previously reported, S. aureus poorly proliferates in the spleen of infected mice. We therefore used the spleen as a non proliferative control. The bacterial burden in kidneys and spleens was quantified by plating CFUs at 4 h, 24 h, 48 h and 72 h after infection. We observed no significant differences between the bacterial counts in the spleens of mice infected either with WT, the Δtkt mutant or the Cp-tkt strain. In each case, a progressive reduction of the bacterial burden was recorded (ranging from 2.5x10 5 , 1.1x10 5 and 1.5x10 5 at 4 h; down to 3x10 3 , 7.3x10 2 and 1.2x10 2 at 72 h, for WT, ∆tkt and Cp-tkt respectively). In contrast, in kidneys, whereas the WT and the Cp-tkt strains readily proliferated (from 3.9x10 4 ± 8.6x10 3 WT and 2.1x10 4 ± 4.6x10 2 Cp-tkt S. aureus per organ at 4 hrs to 7.7x10 7 ± 3.3x10 7 WT and 3.6x10 7 ± 1.3x10 7 Cp-tkt S. aureus per organ at 72 hrs), multiplication of the Δtkt mutant was severely impaired at all time-points tested, with a 10 4 -fold reduction of ∆tkt counts at 24h compared to WT counts (Figure 4). This result confirms the importance of TKT -and most likely of the PPPin bacterial proliferation in vivo and especially during the first 24 hours after infection. This result is consistent with the previously described role of SigB in intracellular growth in vivo [START_REF] Tuchscherr | Staphylococcus aureus Regulator Sigma B is Important to Develop Chronic Infections in Hematogenous Murine Osteomyelitis Model[END_REF].

The SigB pathway may be controlled by modulating carbon source availability.

Inside the cytoplasm of host cells, the Δtkt mutant is likely to deal simultaneously with PPP blocking and altered levels of carbon-based nutrients compared to the outside compartment. The above results suggested that the PPP might take control of rpiRc and sigB expression when bacteria invade endothelial cells. We thus hypothesized that PPP-associated carbon sources may possibly influence rpiRc and/or sigB expression in vitro. We therefore grew WT USA300 in CDM supplemented either with glucose (CDM-glucose), ribose (CDMribose) or fructose (CDM-fructose) a carbon source possibly fuelling the PPP) and quantified by qRT-PCR the transcription of sigB, rpiRc, rsbU/W and asp23 genes. Remarkably, we found that the regulation of the whole sigB operon was impacted by the carbon source used in CDM (Figure 5). Indeed, expression of sigB, rsbU/W and rpiRc decreased in CDM-fructose or CDMribose, compared to CDM-glucose. In contrast, asp23 expression was only decreased by 2fold in CDM-fructose and increased by 3-fold in CDM-ribose, suggesting that in this growth conditions, asp23 expression might be regulated by other factor than sigB (Figure 5). Hence, the availability of carbon sources, and especially those related to PPP, may be sensed by the bacteria and affect the regulation of rpiRc and sigB expression.

Discussion

Inactivation of the tkt gene, encoding the unique transketolase of S. aureus USA300, led to a major decrease in early intracellular proliferation and impaired in vivo the multiplication in a kidney colonization model. Our results suggest that the intracellular proliferation defect of the ∆tkt mutant occurs through the inhibition of sigB and rpiRc expression and the concomitant increased RNAIII transcription, thus highlighting a novel connection between the PPP and these master regulators during cell invasion.

Our whole cell metabolomic analysis of planktonic-grown bacteria (Figure 1, Supplementary Figure S2 and Table S1) showed a huge increase in R5P intermediates in the ∆tkt mutant compared to WT that could be explained by the impaired entry of R5P into glycolysis and a concomitant significant decrease in SH7P relative concentration. In S. aureus, ribose is imported by the RbsU transporter and converted to R5P by the RbsD/RbsK pathway.

RbsR, the repressor of rbsUDK operon, is itself highly regulated by SigB [START_REF] Lei | RbsR Activates Capsule but Represses the rbsUDK Operon in Staphylococcus aureus[END_REF]. Interestingly, S.

aureus grown in CDM-ribose showed a decreased expression of sigB. Hence, stress signals that modulate SigB activity are likely to be important effectors for controlling the quantity of RbsR, thereby affecting ribose uptake.

Significant changes in the relative concentrations of TCA cycle intermediates were also recorded in the ∆tkt mutant compared to WT (Supplementary Figure S3). The TCA cycle plays a central role in maintaining the bacterial metabolic status and has been repeatedly implicated in the regulation of staphylococcal virulence [START_REF] Somerville | Synthesis and deformylation of Staphylococcus aureus delta-toxin are linked to tricarboxylic acid cycle activity[END_REF][START_REF] Ding | Metabolic sensor governing bacterial virulence in Staphylococcus aureus[END_REF]. Hence, changes in TCA cycle activity are likely to induce alterations of the overall metabolome of the bacterium that, in turn, may modulate the activity of metabolite-responsive global regulators such as SigB or RpiR.

RpiRc, a RpiR family-member controlling enzymes involved in sugar catabolism in S.

aureus, has been shown to repress RNAIII [START_REF] Zhu | RpiR homologues may link Staphylococcus aureus RNAIII synthesis and pentose phosphate pathway regulation[END_REF], supporting the notion of a direct connection between the PPP and the regulation of S. aureus virulence. The expression of sigB has also been shown to play a crucial role during the intracellular life of bacteria possibly by downregulating pro-inflammatory virulence factors and increasing the expression of factors promoting persistence [START_REF] Tuchscherr | Staphylococcus aureus dynamically adapts global regulators and virulence factor expression in the course from acute to chronic infection[END_REF][START_REF] Tuchscherr | Sigma Factor SigB Is Crucial to Mediate Staphylococcus aureus Adaptation during Chronic Infections[END_REF].

We propose (Figure 6) that in wild-type S. aureus, transketolase activity results in a positive regulation of rpiRc and sigB transcription and allows the repression of RNAIII.

Consequently, repression of toxins production favours intracellular growth. In contrast, in a ∆tkt mutant, rpiRc and sigB transcriptions are not activated, possibly due to metabolic changes (such as R5P accumulation) and RNAIII is not repressed. At this stage, it cannot be excluded that the enzyme itself might also directly affect transcription of these regulators.

TKT inhibitors are currently actively tested in cancer therapy [START_REF] Xu | Transketolase counteracts oxidative stress to drive cancer development[END_REF] and TKT could constitute an efficient target against tuberculosis [START_REF] Fullam | Structure and function of the transketolase from Mycobacterium tuberculosis and comparison with the human enzyme[END_REF] and malaria [START_REF] Thota | Drug Discovery and Development of Antimalarial Agents: Recent Advances[END_REF]. The present work, highlighting an unprecedentedly reported role of transketolase in S. aureus intracellular survival, suggests that modulation of the pentose phosphate pathway activity may also represent an interesting mean to fight S. aureus infections.

Legends Figure 1. Phenotypic characterization of the ∆tkt mutant

A. Growth of USA300 wild type (WT), tkt mutant (tkt) and its complemented derivative (Cp-tkt). USA300 WT, ∆tkt and Cp-tkt strains were grown in BHI Broth or DMEM for 21 hours at 37°C. Optic densities were measured every hour from 0 to 8 hours and then at 21 hours.

Each panel is a representative experiment.

B. Metabolomic analysis of the ∆tkt mutant strain. Quantitative metabolomic analysis was performed by ion chromatography and tandem mass spectrometry (IC-MS/MS) after overnight cultures, in BHI Broth, from WT USA300 and ∆tkt mutant strains (see Table S1). The heatmap, obtained using MetaboAnalyst (https://www.metaboanalyst.ca), illustrates the metabolic shift between the two strains. Gentamicin concentration of 50µg/ml was maintained throughout the experiment. Images were acquired every 30 minutes using Incucyte ® S3 live cell imaging system.

Leu 662A Quantitative metabolomic analysis was performed by ion chromatography and tandem mass spectrometry (IC-MS/MS) after overnight cultures, in BHI Broth, from WT USA300 and ∆tkt mutant strains (see Table S1). A, B and C. Highlight on metabolic pathways. Light blue: TKT enzymatic reaction. Green arrow: increased concentration in ∆tkt mutant compared to WT strain. Red arrow: decreased concentration in ∆tkt mutant compared to WT strain.

Figure S4

LAMP-1 colocalization assay n.s.

A

Figure S4. LAMP-1 colocalization assay

LAMP-1 colocalization assay. Endothelial EA.hy926 cells were infected with GFP-expressing WT and Dtkt mutant strains. One hour after infection, cells were washed several times with gentamicin containing medium to eliminate extracellular bacteria. A gentamicin concentration of 50µg/ml was maintained throughout the experiment. At 1 hour and 24 hours after infection cells were fixed using 4% PFA and processed for immunohistochemistry. Lysosomes were stained using anti-LAMP1 antibodies and images were acquired by confocal microscopy. Quantification of colocalization between GFP expressing USA300 WT or ∆tkt bacteria and LAMP-1 staining was obtained using ImageJ software. Data were obtained from more than 200 infected cells for each condition and expressed as % of bacteria associated with LAMP-1 ± SD. (ANOVA; n.s.: not statistically different; * p<0.05).

Figure S5. Transcription profile of intracellular S. aureus.

Endothelial EA.hy926 cells were infected with USA300 WT, ∆tkt and Cp-tkt strains as described in figure 2. One, three or seven days after infection cells were washed and collected. RNA from intracellular S. aureus were prepared and gene expression of RNAIII, rpiRc and sigB was analysed by quantitative RT-PCR. Gene expression level was normalized by that of gyrA at days 1, 3 and 7 after infection of EA-hy296 cells. Data are shown as mean normalized expression ± SD of triplicate measurements from two independent experiments. (ANOVA, ns : not statistically different; * p<0.01, ** p<0.001). Experiments presented in Figure S5 were independent of those presented in Figure 3. S. aureus. The epidemic clone S. aureus USA300-LAC (designated USA300-WT) was provided by the Biodefense and Emerging Infections Research Resources (BEI). The GFPexpressing strain was generated by curing the p03 plasmid from USA300-WT and introducing the pCN57-GFP recombinant plasmid (obtained from the BEI) by electroporation as described before [1]. The tkt mutant strain and the complemented strain were constructed as described below. Strains and plasmids used are summarized at the end of these supplementary methods.

Before experiments bacteria were grown 24 hours at 37°C on BHI agar plates supplemented with antibiotics when required and then overnight in BHI Broth from an optical density 600 nm (OD600) of 0.05. The day of experiment, bacteria were resuspended in BHI broth to an OD600 of 0.05 and grown at 37°C until the culture reached the initial-middle log phase (OD600 of 0.6).

Growth curve were performed in brain heart infusion, DMEM and chemically defined medium (CDM) supplemented with ribose or fructose or glycerol when it was required [2].

Internalization rate and survival inside the EA.hy926 endothelial cell line were similar between the GFP strain and the parental strain.

Infection.

When fresh cultures reached OD600 of 0.6, bacteria were added to endothelial cells at a multiplicity of infection (MOI) of 1 and placed in a humidified 5 % CO2 atmosphere at 37 °C for 1 h. One hour after infection, each well was washed three times with 100 µL of phosphate buffer saline (PBS) containing 300 µg/mL gentamicin, to remove extracellular bacteria. Cells were then incubated with 100 µL of cell culture medium containing 50 µg.mL -1 gentamicin.

Infected cells were kept in a humidified 5 % CO2 atmosphere at 37 °C. Importantly, gentamicin is an antibiotic with a high bactericidal effect on S. aureus USA300 (CMI=2 µg.mL -1 ) and a very poor penetration inside eukaryotic cells. In our model the use of gentamicin in the extracellular medium was necessary to abolish extracellular proliferation.

Construction of the S. aureus ∆tkt chromosomal deletion mutant

We inactivated the tkt gene in wild-type S. aureus USA300 strain and substituted it by the kanamycin resistance gene nptII fused with pGro promoter. For this, we used the pMADtemperature-sensitive shuttle vector system [3]. Briefly, the recombinant plasmid pMAD-∆tkt was constructed by overlap PCR. First, the two regions (upstream 694bp, downstream 567bp) flanking tkt and the nptII gene (fused with pGro promoter 1,091bp) were amplified by PCR. The region (2,352 bp) was then amplified by two-step overlap PCR. The resulting PCR product was cloned in pMiniT 2.0 (to yield recombinant plasmid pMiniT/tktUp-npt-tktDown).

This recombinant plasmid was digested with BamHI and EcoRI (New England BioLabs) and the BamHI-EcoRI tktUp-npt-tktDown DNA fragment was finally subcloned into BamHI-EcoRIdigested pMAD. All PCR reactions were realized using Phusion High-Fidelity DNA Polymerase (Thermofisher Scientific) and PCR products were purified using QIAquick PCR purification kit (Qiagen). The pMAD-∆tkt plasmid was first introduced into S. aureus RN4220 prior to electroporation into S. aureus USA300. We used a standard two-step allelic exchange procedure [3] to create the chromosomal S. aureus USA300 ∆tkt strain. The ∆tkt strain was finally checked for loss of the corresponding wild-type genes by PCR sequencing (Eurofins Genomics) using specific primers (described at the end of these supplementary methods).

Construction of the complementation of S. aureus Cp-tkt strain

The plasmid pCN57-CPtkt was constructed by homologous recombination [4]. The pCN57 linear vector and tkt gene fragments were amplified by PCR. To allow recombination, overlapping region of 20pb was added at each ends. PCN57 was linearized using SphI (New England BioLabs). E. coli DH5α were chemically transfected with a mix of both PCR product (insert: vector stoichiometry 2:1, 100ng of vector) in 10 µl MilliQ water. The colonies containing plasmid pCN57-CP-tkt were checked by PCR and the construct was sequenced.

The pCN57-CP-tkt vector was then transfected into S. aureus RN4220 prior to electroporation into S. aureus USA300 ∆tkt. The ∆tkt-Cp-tkt (Cp-tkt) strain was finally checked for complementation of wild-type genes by PCR sequencing (Eurofins Genomics) using specific primers.

Time lapse microscopy

Four days prior to infection, NucLight Red labelled EA.hy926 cells were seeded in ImageLock 96-well plates (Essen BioScience Inc) in culture medium without antibiotic. Cells were infected with wild-type and mutant of the GFP-expressing USA300 strain as described above. Plates were incubated and monitored at 5% CO2 and 37°C for 24 hours in the fully automated microscope Incucyte ® S3 (Essen BioScience). Images were taken every 30 minutes with the 20X objective. The medium was changed once during the observation period. Analysis and time-lapse videos (from which images were extracted) were generated by using Incucyte ® S3 software.

Detailed analysis: Green items were analysed with a threshold of 1 GCU and using Top-hat segmentation mode. Red item were analysed with a threshold of 3 RCU and using Top-hat segmentation mode and edge sensitivity of -25. A spectral unmixing of 10% was applied to remove red signal from green signal. Green item counts were considered as an approximation of the number of intracellular bacteria, and red item counts considered as the number of EA-hy296 cells. (Green items/Red items) represents the number of intracellular bacteria per cell during the time of infection.

Transcriptional analyses

Intracellular bacteria RNA extraction: EA.hy926 cells were grown to confluence in 75 cm 2 flasks and infected with S. aureus as described above. At selected time points, the culture medium was removed and the flasks were washed twice with PBS without antibiotics. Cells were lysed with 5 mL of distilled water for 10 min and the surface of the flask was scraped to detach the cells. The contents were recovered and centrifuged for 5 min at 3000 g. The supernatant was carefully removed and the pellet (which contained cell debris and bacteria) was resuspended in 100 μL of TE buffer containing 20 μg of lysostaphin and incubated for 5 minutes at 37 °C [5] to allow cell wall peptidoglycan degradation.

Planktonic bacteria RNA extraction: S. aureus was resuspended in CDM supplemented with glucose, ribose or fructose to an OD600 of 0.05 and grown overnight at 37°C. Bacteria were centrifuged at 12 000 g for 2 min and incubated in 50 μL of TE buffer containing 10 μg of lysostaphin for 5 minutes at 37 °C. The samples were resuspended in Trizol (Thermofisher Scientific) and treated with chloroform. RNAs were recovered using RNeasy Clean-up column (Qiagen) and DNA was digested using Turbo Dnase (Thermofisher Scientific). RNA concentration was measured using NanoDrop 1000 (Thermofisher Scientific). Reverse transcription was carried out with 500 ng of RNA and 0.5 μg of random primers (Promega). Luna Universal qPCR Master Mix (New England Biolabs) according to the manufacturer's recommendations. Transcript levels were analyzed using a 7900HT Fast Real-Time PCR System (Applied Biosystems) according to the standard settings of the system software. The thermal cycling conditions were: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Gene specific primer sequences are presented at the end of these supplementary methods. Expression of the gene gyrA was chosen as a reference.

We used the "The Relative Standard Curve Method" for analyzing the qRT-PCR data.

Metabolomic analyses

Bacteria: Metabolite profiling of S. aureus isolates was performed by liquid chromatographymass spectrometry (LC-MS) as described [6,7]. Two independent experiments with three biological replicates were performed for each isolate. Briefly, S. aureus strains were grown in BHI Broth overnight to stationary phase and pelleted by centrifugation. Metabolic activity was blocked by immersion in liquid nitrogen for 10 sec. Metabolites were extracted using a solvent mixture of Methanol/ACN/H2O (50:30:20) at -20 °C (Extraction solution). Samples were vortexed for 5 min at 4°C, and then centrifuged at 16,000 g for 15 minutes at 4°C.

Transketolase activity assay

Bacteria were grown to stationary phase in BHI Broth and 1 ml of culture was harvested.

Bacteria were washed in PBS, pelleted by centrifugation, and resuspended in 300 µl of 50 mM Tris with 60 µg lysostaphin, pH 7.5. Samples were incubated at 37 °C for 5 min and then sonicated at 4 °C (30s on, 30s off, repeated 30 times) (Diagenode). Samples were centrifuged at 12,000 g and 4 °C for 10 min to remove cell debris and protein concentration was determined using BCA assay with BSA as a standard (Thermo Fisher Scientific). TKT activity was analysed as described previously [10]. Briefly, each enzymatic reactions were performed at a final volume of 100 µl containing 20 µl of supernatant, 50 mM Tris (pH 7.5), 0.24 mM MgCl2, 10 µM thiamine pyrophosphate, 0.5 mM NADH, 3 units of glycerol-3phosphate dehydrogenase, 10 units of triosephosphate isomerase, 0.5mM D-xylulose-5phosphate, and 0.5mM D-erythrose-4-phosphate (all reagents obtained from Sigma-Aldrich).

The reaction proceeded at 37 °C for 5 min, and the decrease in A340 nm was monitored with a microplate reader (Molecular Devices). Transketolase enzyme activity in bacteria lysates was expressed as units per microgram of total protein. One unit of enzyme was defined as the amount of enzyme that oxidized 1 µmol of NADH per minute. for 20 min and glass coverslips were mounted in Mowiol (Cityfluor Ltd.). Cells were examined using an X63 oil-immersion objective on an Apotome microscope (Zeiss TM ). Colocalization of GFP expressing bacteria with LAMP-1 staining was quantified by using Image J software (plug in: Coloc based Sur-vol _ Beta v6 ). Data were obtained from more than 200 infected cells for each condition and expressed as % of bacteria associated with LAMP-1 ± SD.

LAMP-1 colocalization assay

In vivo infection

For all experiments we used 6 to 10 weeks-old female BALB/c (Janvier Labs) with access to food and water ad libidum.

Infection. Overnight cultures of S. aureus in BHI Broth were diluted in BHI Broth to an OD600 of 0.05. Bacteria were grown at 37°C until the culture reached the initial-middle log phase (OD600=0.6) and resuspended in a sterile isotonic saline solution to an OD600 of 0.4.

Mice were infected with 10 7 bacteria intravenously (iv) in the tail vein. Inoculum titers were checked by spreading dilutions of the preparations on BHI agar plates. For bacterial burden determination, mice were euthanized after 4, 24, 48 and 72 hours. Sequential dilutions of homogenized preparations from spleen and kidney were spread onto BHI agar plates.

Mice infected with the complemented strain were treated with erythromycin to avoid loss of the complementing plasmid. For this, mice received one dose of erythromycin 16 hours before infection and 2 doses of erythromycin per day until they were sacrificed (500 µg of erythromycin per dose). Experiments presented in Figure S5 were independent of those presented in Figure 3.

Statistics

WT tkt

Cp-tkt 0

  5 GFP expressing particles recorded per cell (Figure 2A-B). Active proliferation of WT S. aureus inside EA.hy926 cells was confirmed after 24 hours of infection (Figure 2A-B and Movie 1)

Figure 2 .

 2 Figure 2. Intracellular proliferation and persistence of S. aureus.

Figure 3 .

 3 Figure 3. Transcription profile of intracellular S. aureus.

Figure 4 .

 4 Figure 4. Proliferation of S. aureus in the spleen and the kidney of infected mice.Bacterial loads in the spleen and the kidney of BalB/c mice infected intravenously with 10 7

Figure 5 .

 5 Figure 5. Carbon sources influence the transcription profile of the SigB pathway.

Figure 6 .Movie 1 :

 61 Figure 6. Schematic depiction of the impact of TKT inactivation on sigB and rpiRc

Movie 2 :T r a n s k e t o l a s e a c t i v i t y ( U µ g - 1 )Figure S1 .

 21S1 Figure 1

Figure S3 .

 S3 Figure S3

T im e p o s t in fe c tio n % o f b a c t e r i a a s s o i a c t e d w i t

  Figure S5

  Cell line. EA.hy926 cell line (ATCC CRL-2922), originally derived from human umbilical vein, were grown in Dulbecco's modified Eagle high glucose medium (DMEM, Dominique Dutscher) supplemented with 10 % fetal bovine serum (FBS, Thermofisher scientific) in a humidified 5 % CO2 atmosphere at 37 °C. When indicated cells were incubated with no glucose DMEM supplemented with glucose, ribose or fructose at a concentration of 25mM.To facilitate cell identification and counting during time lapse experiments, EA.hy926 cells were transduced with the IncuCyte ® NucLight Red Lentivirus, following the manufacturer's recommendations, to obtain red nuclear labelling of living cells due to the expression of the red fluorescent protein mKate2 containing a nuclear localization signal.

  After denaturation at 70 °C for 5 min, 15 μL of the mix containing 4 μL M-MLV 5 X reaction buffer, 1 μL of 0.5 mM PCR Nucleotide Mix, 0.5 μL of RNasin Ribonuclease Inhibitor, and 1 μL M-MLV Reverse Transcriptase (Promega) was added. Samples were incubated 5 min at 25 °C, 1 h at 42 °C, then 15 min at 70 °C and chilled on ice. Quantitative RT-PCR was performed using FastStart SYBR Green qPCR Master Mix (ThermoFischer Scientific) or

  Endothelial cells: EA.hy926 endothelial cells were grown to confluence on a 12-well plate in no glucose DMEM supplemented with glucose or fructose or ribose (25 mM). One independent experiment with three biological replicates was performed for each condition. At each time points the cells were washed with cold PBS. Metabolism was blocked by plunging the plate into liquid nitrogen for 10 seconds. The cell metabolites were extracted from the samples by placing the cell culture plate on a rocking shaker at 4°C for 10 minutes. The extraction solution from each well was then collected and centrifuged at 16,000 g for 15 minutes at 4°C. The supernatants were collected and analysed by LC-MS using SeQuant ZIC-pHilic column (Millipore) for the liquid chromatography separation[6]. The aqueous mobile-phase solvent was 20 mM ammonium carbonate plus 0.1% ammonium hydroxide solution and the organic mobile phase was acetonitrile. The metabolites were separated over a linear gradient from 80% organic to 80% aqueous phase for 15 min. The column temperature was 48 °C and the flow rate was 200 μl/min. The metabolites were detected across a mass range of 75-1,000 m/z using the Q-Exactive Plus mass spectrometer (Thermo) at a resolution of 35,000 (at 200 m/z) with electrospray ionization and polarity switching mode. Lock masses were used to insure mass accuracy below 5 ppm. The peak areas of different metabolites were determined using TraceFinder software (Thermo) using the exact mass of the singly charged ion and known retention time on the HPLC column. By this approach we were able to identify and measure: Glucose, Glucose-6P (G6P), UDP-glucose (UDP-Glc), Sedoheptulose-7-P (SH-7P), Ribose 5-phosphate, Glyceraldehyde 3-phosphate, Lactate, Pyruvate, Phospho-Enol-Pyruvate (PEP), Citrate (Cit), cis-aconitate (cis-aco), Succinate (Suc), Fumarate (Fum), Malate (Mal), alpha ketoglutarate (aKG), dihydroxyacetone phosphate (DHAP), N-carbamoyl-L-aspartate, Nicotinamide adenine dinucleotide (NAD), NADH, Nicotinamide adenine dinucleotide phosphate (NADP), NADPH, Adenine, Adenosine, Adenosine Mono, Di and Triphosphate (AMP, ADP, ATP), Cytidine, Cytidine Mono, Di and Tri-phosphate (CMP, CDP, CTP), Uridine Mono, Di and Tri-phosphate (UMP, UDP, UTP), Hypoxanthine, Guanine, Guanaosine, Guanidine Mono and Di-phosphate (GMP, GDP), Inosine monophosphate (IMP), Pantothenate, Dephospho-Coenzyme A, Coenzyme A (CoA), Flavin adenine dinucleotide (FAD), Niacin, Nicotinamide, 3-hydroxybutyrate, Urate, 5-oxo-L-Proline, Acetylaspartate, Acetyl-glutamine, Alanine, Arginine, Argininosuccinate, Asparagine, Aspartate, Betaine, Citrulline, Cystathionine, Cystine, Glutamine, Glutamate, Glycine, Histidine, IsoLeucine, Leicine, Sarcosine, Lysine, Methionine, Ornithine, Phenylalanine, Proline, Serine, Taurine, S-Adenosyl-L-methionine, Threonine, Tryptophan, Tyrosine, Valine, Butyric acid, Carnitine, Decanoic acid, Lauric acid, Hexanoic acid, Linoleic acid, Myristic acid, Octanoic acid, Oleic acid, Palmitic acid. Statistical analyses were performed using MetaboAnalyst 4.0 software [8, 9]. The algorithm for heatmap clustering was based on the Pearson distance measure for similarity and the Ward linkage method for biotype clustering. Metabolites with similar abundance patterns were positioned closer together.

TKTFigure S3 .

 S3 Figure S3

FigureFigure S5 .

 S5 Figure S4

used for ∆tkt mutant construction and complementation Primer name Primer sequence (From 5' to 3')

  Data were analyzed using GraphPad Prism software. Results are presented either with one representative experiment for clarity or as means ± standard deviation (SD). The number of biological and technical replicates is indicated in the legend of each figure. For two-sample comparisons statistical significance was measured using unpaired two-tail Student's t-test.For comparisons between more than two groups, statistical significance was measured using one-way analysis of variance (ANOVA) with Dunnett's correction. P values of p<0.05 were considered to indicate statistical significance. . nptII: neomycin phosphotransferase gene, determing kanamycin resistance.

	pMiniT 2.0		Plasmid used to clone upstream and	New England Biolabs
			downstream regions from tkt gene and
			nptII, Amp R
	Ethics statement
	All experimental procedures involving animals were conducted in accordance with guidelines
	established by the French and European regulations for the care and use of laboratory
	animals (Decree 87-848, 2001-464, 2001-486 and 2001-131 and European Directive 2010/63/UE) and approved by the INSERM Ethics Committee (Authorization Number: 75-906). Primers tktup F: CGGAATTCGAAGTAGGATTAACTCAAGAAGAAGC
		R: TTTAGCTCGACTAATCCATACAACTGCTAAAGCTAATCCTACTGAC
	tktdn Strains and plasmids used in this study. F: CCTTCTTGACGAGTTCTTCTGAGGATGGCATAAATATGTAGGTACTG R: CGGGATCCATTTGCATCATCATCATACGAAGC
	Strain or plasmid nptII F: TTGTATGGATTAGTCGAGCTAAA Relevant characteristic(s)		Source of reference
	Strains	R: TCAGAAGAACTCGTCAAGAAGG
	E.coli s_tkt	F: GAATATCGAAAGAATTAACGAGTTAGC
	DH5α	Φ80' lacZ ∆M15 gyrA96 recA1 relA1 R: CGCACTCTTCATATTTTGATCCAT	Invitrogen
	s_pMAD	endA1 thi-1 hsdR17 F: TCTGGCCATTGCTCTGGGTTA
	R: GCTGTCCCTGATGGTCGTCAT USA300 LAC S.aureus MRSA containing phage ΦSA2usa (PVL), Ery R HRtkt F: CTTTGAGTGAGCTGGCGGCCGGATCCGGGTTTAAACAACAAATTGAAAATAC BEI Resources R: CATAGTAAGCTGCATGCAGCCCCGGGTTATAAGCTCATAACTTGGTTTAAGATATTTTC
	USA300-Ery S HRpCN57 F: ACCAAGTTATGAGCTTATAACCCGGGGCTGCATGCAGCTTACTATG Erythromycin-sensitive derivative of Laboratory collection
	RN4220	USA300 Restriction-deficient transformation R: TCAATTTGTTGTTTAAACCCGGATCCGGCCGCCAGCTCACTCAAAG Laboratory collection
			recipient S. aureus
	USA300 ∆tkt	tkt defective mutant, Kan R		This study
	USA300 ∆tkt-CPtkt	Complemented strain, USA300 ∆tkt	This study
			containing pCN57-CPtkt, Ery R Kan R
	USA300 LAC-gfp	Strain containing pCN57-GFP, Ery R	Laboratory collection
	USA300 ∆tkt-gfp	USA300 ∆tkt containing pCN57, Ery R	This study
			Kan R
	Plasmids
	pMAD		Plasmid with a thermosensitive origin,	Laboratory collection
			used for gene deletion; Amp R Ery R
	pMAD-∆tkt		Derivative of pMAD with upstream and	This study
			downstream sequences from tkt gene
			and nptII a , Amp R Ery R Kan R
	pCN57		Plasmid containing gfp expression	Laboratory collection
			cassette (constitutive expression),
			Ery R
	pXT02		Derivative	of	pCN57.	The	This study
			gfpexpression cassette was replaced
			by the sequence of the tkt gene with
			its own promoter.

a

Primers used for RT-qPCR.

  Figure S2. Transketolase proteins domain organization and structure. A. Conserved domains found in S. aureus transketolase. Transketolase_N (PF00456 domain): Transketolase, thiamine diphosphate binding domain; Transket_pyr (PF02779 domain): Transketolase, pyrimidine binding domain; Transketolase_C (PF02780): Transketolase, C-terminal domain (putative regulatory molecule binding site). B. Protein model of S. aureus transketolase. Swiss-Model was used to predict the structure of S. aureus TKT (SAUSA300_1239) as a homodimer based on the template 3M49.1.A (Crystal Structure of Transketolase from Bacillus anthracis). The QMQE score and QMEAN were 0.83 and -0.59 respectively. C. Table showing coverage percentage between transketolase amino acid sequences and percentage of identical amino acids between aligned transketolase amino acid sequences. Protein sequences were retrieved from UniProt database and aligned using Clustal Omega.

	asp23 Figure S2		F: TCGCTGCACGTGAAGTTAAA
	A	1		R: CACCAACTTCAACAGATACACC	662 aa
		I			I
			Transketolase_N	Transket_pyr	Transketolase_C
	B	Phe 2A
	C				Leu 662A
		Protein ID Protein ID	% coverage % identity Species % coverage % identity Species
		TKT_HUMAN	100.0%	100.0% Homo sapiens
		TKT_MOUSE	100.0%	94.7%	Mus musculus
		TKT2_ECOLI	94.5%	24.1%	Escherichia coli
		TKT_HAEIN	94.2%	22.9%	Haemophilus influenzae
		TKT1_VIBCH	94.4%	24.0%	Vibrio cholerae
		TKT_DICDI	94.2%	22.6%	Dictyostelium discoideum
		TKT1_YEAST	94.5%	23.8%	Saccharomyces cerevisiae
		TKT_TREPA	93.9%	21.9%	Treponema pallidum
		TKT_MYCBO	96.6%	20.6%	Mycobacterium bovis
	Gene name TKT_STRPN	94.2%	Primer sequence (From 5' to 3') 23.0% Streptococcus pneumoniae
	RNAIII TKT_STAAS	94.4%	F: GCCATCCCAACTTAATAACCATGTAAAATTAGC 22.4% Staphylococcus aureus
		TKT_BACSU	94.9%	R: GCGATGTTGTTTACGATAGCTTACATGCTAG 22.7% Bacillus subtilis
	rpirC		F: CATACACGTACTACACGCGCACTTAATC
				R: GTCGGTAGCAACTACAAAAGATGCG
	tkt			F: GGTCATAATGGTGCATCTCGTGC
				R: CATTGGATTTGTTTGAACCAGCAAGG
	gyrA		F: GGTGGATTTGAAAACTTAGAGGACGAAGAC
				R: CATACCTTGAACACCACGACCACC
	sigB		F: GGTGCCATAAATAGATTCGATATGTCCTT
				R: CTTTTGATTTCACCGATTACAGTAGGTACT
	rsbU		F: CGCGTGAAGATGTGTTCAAGAC
				R: CTATCTCTTTATCGTGAACTTGAAG
	rsbW		F: GAAGATTTTATCGAAATGCGC
				R: CATCATATGTAGCACCAGCTCTC

Acknowledgements

We are grateful to the Cell imaging core facility of the "Structure Fédérative de Recherche" Necker INSERM US24/CNRS UMS3633 for his technical support. The following reagents were provided by the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for distribution by BEI Resources, NIAID, NIH: Staphylococcus aureus, Strain USA300-0114, NR-46070; Escherichia coli -Staphylococcus aureus Shuttle Vector pNR-46158, Recombinant in Staphylococcus aureus, NR-46158.

These studies were supported by INSERM, CNRS and Université Paris Descartes Paris Cité Sorbonne. This work was supported by Vaincre La Mucoviscidose (grant RF20180502254). Xin Tan was funded by a scholarship from the China Scholarship Council (n° CSC NO. 201508500097). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Ethics statement

All experimental procedures involving animals were conducted in accordance with guidelines established by the French and European regulations for the care and use of laboratory animals C. Growth of USA300 wild type (WT), ∆tkt mutant (∆tkt) and its complemented derivative (Cptkt). USA300 WT, ∆tkt and Cp-tkt strains were grown in CDM supplemented with ribose as sole carbon source for 21 hours at 37°C. Optic densities were measured every-hour from 0 to 8 hours and then at 21 hours. Each panel is a representative experiment.

Results

)


Keywords: Degradation, dilute nitrides, electrons, InGaAsN, irradiation, MJSC, solar cell, DLTS, PL, EQE

Degradation of InGaAsN pin subcell under 1 MeV electron irradiation was studied by characterizing solar cells and dilute nitride bulk layers before and after irradiation. The measurements show that these cells retain more than 94% of their original photocurrent after an 10 15 cm -2 electron-irradiation. Moreover, no significant degradation of the optoelectronic properties is observed after irradiation.

I. INTRODUCTION

Multi-junction solar cells (MJSC) based on III-V materials offer today the highest conversion efficiency of all solar technologies, as their structure leads to the optimal usage of the solar spectrum. MJSC are then of prime interest for space applications where delivered power over charge ratiois the key parameter. Today's space solar cell market is dominated by the GaInP/(In)GaAs/Ge tri-junction cell lattice-matched to its substrate, with 28-30 % efficiency [START_REF] Bett | Overview about technology perspectives for high efficiency solar cells for space and terrestrial applications[END_REF]. Both the architecture and the materials of this standard tri-junction cell have already been optimized and room for improvement is now extremely limited.

Changing or adding subcells appears then to be necessary in order to overpass the 30 % efficiency level. Theoretical calculations with the optimal bandgap combination point out the need to develop a 1 eV subcell to improve the spectral overlapping [START_REF] Kurtz | Projected performance of threeand four-junction devices using GaAs and GaInP[END_REF]. One way to improve the structure is to subtstitute the Ge for a 1 eV material in the bottom cell. Another possibility is to fabricate a 4-junction solar cell by introducing a 1 eV subcell between the GaAs and the Ge ones.

A major requirement for the 1 eV subcell is that the material needs to be lattice-matched on GaAs or Ge, insuring the best crystalline quality. Dilute nitride (In x Ga 1-x As 1-y N y ) alloys have been proposed as candidates for this application as both their bandgap and their lattice parameter can be tuned by varying the indium and nitrogen contents [START_REF] Kondow | GaInNAs: A Novel Material for Long-Wavelength-Range Laser Diodes with Excellent High-Temperature Performance[END_REF]- [START_REF] Geisz | III-N-V semiconductors for solar photovoltaic applications[END_REF]. As a notable proofof-concept, Solar Junction reported in 2011 a world record efficiency (at the time) of 43.5 % under concentration for a trijunction incorporating an InGaAsN-based bottom cell [START_REF] Wiemer | 43.5% efficient lattice matched solar cells[END_REF].

Furthermore, space applications require the subcell to be radiation hard since constant irradiation inevitably leads to cell degradation. This degradation can be particularly problematic for MJSC since its subcells are connected in series and thus Several degradation studies have already been reported for InGaAsN cells but the irradiations were carried out on nonoptimized cells (regarding both the material and the architecture) grown by metal organic vapor phase epitaxy (MOVPE) [START_REF] Kurtz | 1-MeV-electron irradiation of GaInAsN cells[END_REF]- [START_REF] Khan | DLTS analysis of radiation-induced defects in InGaAsN solar cell structures[END_REF]. More recent studies reported on quasioptimized structures achieving short-circuit current density (J sc ) higher than 15 mA/cm² under integrated illumination conditions (AM0 > 870 nm [START_REF] Polojarvi | Influence of As/group-III flux ratio on defects formation and photovoltaic performance of GaInNAs solar cells[END_REF] and AM1.5 > 830 nm [START_REF] Wilkins | Design of thin InGaAsN(Sb) n-i-p junctions for use in four-junction concentrating photovoltaic devices[END_REF]). These cells are based on a n-i-p design which strongly relies on a field-aided collection regime. Moreover, the dilute nitride material quality was improved by using molecular beam epitaxy (MBE) instead of MOVPE, leading to lower background carrier concentration.

As lattice-matching and current-matching conditions can be fulfilled, InGaAsN subcell integration into MJSC is feasible [START_REF] Wiemer | 43.5% efficient lattice matched solar cells[END_REF], [START_REF] Tukiainen | High-efficiency GaInP/GaAs/GaInNAs solar cells grown by combined MBE-MOCVD technique[END_REF]. It is now crucial to quantify and understand the degradation of InGaAsN under standard irradiation conditions. To do so, we propose to study the InGaAsN subcell degradation by combining the current-voltage characterization (I-V), the external quantum efficiency (EQE), the deep level transient spectroscopy (DLTS) and the photoluminescence (PL) at the beginning of life (BOL) and after samples irradiation with a 1 MeV electrons beam.

II. EXPERIMENTAL METHOD

A. InGaAsN cell and diode fabrication for DLTS characterization

The InGaAsN p-i-n cell structure is grown by MBE on a 4inch n-GaAs (001) substrates. The device structure is composed of a p-i-n GaAs/InGaAsN/GaAs junction and include a n-Al 0.4 Ga 0.6 As back surface field (BSF), a p-Al 0.4 Ga 0.6 As window layer capped with a p + GaAs layer (Fig. 1a). TABLE I. summarizes the InGaAsN growth conditions of this series of studied cells, labelled as A, B, C and D. Other growth parameters were kept constant. The composition of the InGaAsN active layer was set to 4.5 % for In and ≈ 1.6 % for N corresponding to the lattice-matched condition. No rapid thermal processing (RTP) was performed on these wafers.

Furthermore, a 1.5 µm-thick non-intentionally doped (NID) InGaAsN bulklayers are grown on 4-inch n-GaAs (001) substrate with different As/III ratios for PL and DLTS characterizations. The growth conditions of these samples (E and F) can be found in TABLE I. Some of these samples were annealed at 750 °C for 30 s before being processed while others were processed as-grown. Electrochemical voltage capacitance measurements show that the InGaAsN layers are n-doped with concentrations ranging from 3.10 16 to 1.10 17 cm - 3 . AuGeNi/Au metallization was carried out on the backside surface of the substrate by sputtering deposition and a Ti/Au metallic grid was deposited on the surface front by e-beam evaporation followed by a lift-off step. Wafers were then annealed at 350 °C during 90 s in order to achieve good ohmic contacts. Wet etching was performed using H 3 PO 4 /H 2 O 2 to form mesa structures to isolate each cell. The GaAs cap layer was afterwards selectively removed using C 6 H 8 O 7 /H 2 O 2 . No anti-reflective coating (ARC) was deposited. Wafers were finally cleaved in 0.25 cm² and 1 cm² cells respectively for EQE and I-V measurements, with grid densities ranging from 0 to 10 %. The processed cell structure cut view is shown in Fig. 1b.

The InGaAsN bulk layers were processed with the same technological steps as the solar cells in order to fabricate Schottky diodes (NID InGaAsN / TiAu) for DLTS measurements.

B. Irradiation conditions

Cells and samples A to F were irradiated in the MIRAGE facility at ONERA with a Van de Graaff accelerator providing 1 MeV electron beam [START_REF] Duzellier | AXEL lab.: representative ground simulation for investigating radiation effects in materials and electronics[END_REF]. The electron flux was maintained below 5.10 10 e -/cm 2 /s (≈ 8 nA/cm²) in order to to avoid irradiation-induced overheating as samples were not thermally regulated during irradiation. However, they were mounted on aluminum nitride support and copper plate (see Fig. 2), which both have high thermal conductivity. The irradiation chamber was at room temperature and the pressure was set tbelow 10 - 6 mbar.

Four solar cells thereafterlabelled A1, B1, D1 and G1 were irradiated four times, with cumulative fluences of respectively 5.10 13 , 1.10 14 , 5.10 14 and 1.10 15 e -/cm 2 , allowing us to measure their I-V characteristics at various fluence levels. The rest of the samples was irradiated in one step up to 1.10 15 e -/cm². GaAs p-n cells were irradiated as degradation references (G cells).

III. RESULTS AND DISCUSSION

A. InGaAsN solar cells response to irradiation

The I-V characteristics were measured with an Oriel solar simulator equipped with a Xenon lamp and a AM0 filter, preliminary calibrated with a reference tri-junction cell from Azur Space. Fig. 3a shows the J-V characteristics of the GaAs reference before and after the different steps of irradiation. As commonly reported, J sc decreases because of defect introduction which shortens the minority carrier diffusion length leading to a reduction of the collection efficiency [START_REF] Warner | Correlation of Electron Radiation Induced-Damage in GaAs Solar Cells[END_REF], [START_REF] Danilchenko | 1MeV electron irradiation influence on GaAs solar cell performance[END_REF]. The normalized J sc values are plotted as a function of the fluence in Fig. 3b. A degradation can be observed as the fluence increases, with a degradation rate in good agreement with the results reported in the literature [reference]. Moreover, the J sc degradation is found to be consistent with the EQE measurements (not shown here). Open-circuit voltage (V oc ) falls from 0.885 to 0.845 V accounting for 4.5 % degradation. This degradation correlates with an increase of one order of magnitude of the dark current density and is consistent with already published results [START_REF] Warner | Correlation of Electron Radiation Induced-Damage in GaAs Solar Cells[END_REF], [START_REF]GaAs Solar Cell Radiation Handbook[END_REF]. The J-V curves of the InGaAsN cells were measured under spectrally-filtered light in order to reproduce the absorption of upper subcells in a MJSC. For that purpose, a Newport highpass filter (λ > 870 nm) was placed within the optical path. Fig. 4a presents the evolution of the J-V curves of the A1 cell for different irradiation levels. The B1 and D1 cells exhibited the same J-V variation vs fluence as the A1 cell. Electron irradiation was found to be less detrimental to the InGaAsN cells than to the GaAs reference cell (G1), since the mean degradation of J sc and V oc after 10 15 e -/cm² were lower than 6 % and 2 %, respectively. The degradations of J sc for all dilute nitride cells are plotted in Fig. 4b. A clear difference in degradation rate can be observed between the cells irradiated directly at 1.10 15 e -/cm² and the cells subjected to successive irradiations. That could be due to defect annealing (as discussed below) enhanced by uninterrupted irradiation. Otherwise, epitaxial growth conditions were not found to affect the degradation behavior of the cells, as J sc and V oc degradations for A, B, C and D were found to be roughly the same. We performed EQE measurements on solar cells directly irradiated at 10 15 e -/cm². As it can be seen on the Fig. 5, irradiation has virtually no effects on the spectral response of InGaAsN cells and it can even be observed that the EQE signal in the range 650-1050 nm slightly increases after irradiation for cells A and B. However, this improvement disappears for longer wavelength which explains why it does not lead to higher J sc under filtered AM0 light.

To describe the radiation hardness exhibited by InGaAsN cells, we propose three hypotheses. First, defect annealing and defect introduction occur simultaneously under irradiation. Indeed, annealing of growth defects during irradiation have already been reported for GaAs [START_REF] Pons | Irradiation-induced defects in GaAs[END_REF] and InGaAsN [START_REF] Pavelescu | Effects of 7-MeV electron irradiation on photoluminescence from 1-eV GaInNAs-on-GaAs epilayers[END_REF]. This phenomenon is based on the recombination-enhanced annealing mechanism coupled with the creation of vacancies in the lattice. Irradiation induces multiple ionizations in the material which in turn lead to a high carrier recombination rate. Carrier recombination can then locally bring enough energy to the lattice to anneal defects. However, for the defect recombination to be possible, vacancies need to be present in the lattice as it greatly enhances atomic diffusion [START_REF] Bourgoin | Enhanced diffusion mechanisms[END_REF]. Furthermore, the atomic diffusion of defects increases with temperature, which could explain why direct irradiation leads to lower degradation, as the sample might heat up more than it does under cumulative irradiation. The second hypothesis to explain the radiation hardness of the cells is the predominance of growth defects over the irradiation-induced ones. Indeed, if at BOL the defect concentration is already high, then the contribution of the defects created during irradiation on the minority carrier diffusion length remain marginalprovided a low defect introduction rateand would have a negligible impact on the cell's properties. This hypothesis is supported by the fact that a high density of N-related defects may be present in some dilute nitride materials, arising from the small size and the high electronegativity of the nitrogen anion [START_REF] Polojarvi | Influence of As/group-III flux ratio on defects formation and photovoltaic performance of GaInNAs solar cells[END_REF], [START_REF] Kaplar | Deep levels in pand n-type InGaAsN for high-efficiency multi-junction III-V solar cells[END_REF], [START_REF] Khan | Correlation of nitrogen related traps in InGaAsN with solar cell properties[END_REF]. This latter hypothesis underlies that the optical quality would be strongly affected, which does not seem to be the case with regard to the strong PL and EQE signals observed with our samples.

A third way to explain this radiation tolerance comes from the pin structure and the collection regime of the cells. As we said previously, the InGaAsN pin cells rely on the built-in electric field extending across the whole intrinsic layer to collect carriers, as a workaround for dilute nitride's low minority carrier diffusion length. Consequently, the decrease of this diffusion length through the introduction of defects has a smaller impact on the InGaAsN cells performance than it has on GaAs ones. A low J sc degradation for GaAs pin solar cells has already been reported and attributed to this drift regime [START_REF] Ohshima | Change in the electrical performance of GaAs solar cells with InGaAs quantum dot layers by electron irradiation[END_REF].

B. DLTS and PL evolution with irradiation

To assess the defect annealing explanation, i-DLTS measurements were performed on InGaAsN samples before and after 1 MeV electron irradiation. A Phystech DL8000 spectrometer and its analysis software were used for that matter. The reverse voltage was set to -1 V and we used a 0.5 ms filling pulse of 0 V. We measured the current transient between 10 and 50 ms after the end of the pulse. The DLTS spectra of as-grown and annealed samples from E and F wafers are shown in Fig. 6a. Although the crystal quality is different from one BOL sample to another -as depicted by the DLTS spectra -there is no significant difference between BOL and corresponding irradiated samples. Fig. 6b shows the deconvolution of two emission peaks that reveal the presence of two electron traps, with activation energy approximately equal to 0.6 and 0.85 eV (as determined with the Arrhenius plot in Fig. 6c). As the bandgap energy of the DLTS sample is equal to 1.05 eV, the 0.6 eV trap appears to be quite deep and probably acts as an effective non-radiative recombination center. This defect is conjectured to be a nitrogen split interstitial (N-N) As since it is a typical defect in dilute nitrides and as its activation energy was theoretically calculated to be 0.66 eV below the conduction band [START_REF] Zhang | Nitrogen solubility and induced defect complexes in epitaxial GaAs:N[END_REF]. Interestingly, as the amplitude of the DLTS signal is proportional to the defect concentration, it can be deduced that irradiation does not lead to a change of any of these defects concentration. This result implies that the radiation hardness exhibited by the InGaAsN cells cannot be explained by the annealing of deep level growth defects such as (N-N) As . However, it also shows that there is no sensible introduction of deep-level defects through the irradiation process, which supports our second hypothesis.

PL measurements were then made to assess the impact of irradiation on optical properties, as depicted in Fig. 7. The PL spectra were obtained at 300K with a 455 nm laser acting as the excitation source with a 15 mW/cm² power density. From Fig. 7 it can be seen that the PL signal increases after irradiation for as-grown samples whereas it remains constant or degrades for annealed ones. The fact that the PL increases after irradiation for as-grown samples means that there is less non-radiative recombination after irradiation. It points out the presence of growth-related shallow defects (not detectable with DLTS) and their curing during the irradiation process. This result correlates well with the slight increase of the EQE of unannealed cells after irradiation (Fig. 5). Additionally, we can see that the PL intensity of unannealed samples after irradiation gets closer to the intensity of annealed samples at BOL. This suggests that the irradiation acts as the 750 °C RTP for curing shallow defects without however leading to the characteristic bandgap blueshift associated with thermal annealing of dilute nitrides. The plateau-like degradation of the InGaAsN cells between 10 14 and 10 15 e -/cm² as well as the small J sc recovery of B1 and D1 cells could then results from the annealing of these shallow defects. Moreover, the fact that the PL signal of the annealed samples does not increase (and even decreases in the case of wafer F) after irradiation can be explained by the change of InGaAsN preferential atomic bonding after RTP. Indeed, nitrogen atoms are reported to go from a Ga-N bonding configuration to a In-N one after annealing [START_REF] Volz | Optimization of annealing conditions of (GaIn)(NAs) for solar cell applications[END_REF], [START_REF] Uno | Thermal annealing effects and local atomic configurations in GaInNAs thin films[END_REF], which could affect the defect energy formation and consequently the radiation hardness of the material.

IV. CONCLUSION

InGaAsN pin subcells and bulk layers were fabricated, electron-irradiated and characterized, showing a better resistance to 1 MeV electrons than GaAs cells. We have suggested three physical interpretations to explain this degree of tolerance based on both the specific material properties and the junction structure. DLTS and PL analysis on InGaAsN bulklayers show that the radiation hardness cannot be explained by the introduction and the annealing of deep-level defects. Indeed, the growth-defect density may be too important compared to the density of irradiation-induced defects. The PL enhancement and the slight EQE increase are then attributed to the annealing of shallow defects during irradiation.

Fig. 1

 1 Fig. 1 Structure of the epitaxial stack of the InGaAsN cell (a) and cut view after processing steps (b)

Fig. 2

 2 Fig. 2 Cells and samples for PL mounted on their holders for irradiation

Fig. 3 a

 3 Fig. 3 a) J-V characteristics of GaAs G1 cell under AM0 light at 25°C and for different fluences b) Degradation of the photocurrent for G1 as a function of fluence, compared to literature data[START_REF] Warner | Correlation of Electron Radiation Induced-Damage in GaAs Solar Cells[END_REF],[START_REF] Danilchenko | 1MeV electron irradiation influence on GaAs solar cell performance[END_REF]

Fig. 4 a

 4 Fig. 4 a) J-V characteristics of InGaAsN A1 cell under AM0 > 870 nm light at 25°C and for different fluences b) Degradation of the photocurrent in InGaAsN cells as a function of fluence

Fig. 5

 5 Fig. 5 EQE before and after 1 MeV electron irradiation for 4 InGaAsN cells

Fig. 6 a

 6 Fig. 6 a) DLTS spectra of samples before and after 1 MeV electron irradiation b) Deconvolution of the DLTS spectra of sample E As-grown-BOL in 2 emission peaks c) Arrhenius plot of E1 and E2 traps.

Fig. 7

 7 Fig. 7 PL spectra of samples before and after 1 MeV electron irradiation

  France each cell is required to generate the same amount of photocurrent.

1

(1) M. Levillayer, S. Duzellier, T. Nuns, C. Inguimbert, T. Le Cocq, R. Rey, C. Pons and L. Artola are with ONERA/DPHY, Université de Toulouse, 2 av. E. Belin, 31055 Toulouse, France (e-mail: maxime.levillayer@onera.fr). (2) F. Olivié, I. Massiot, A. Arnoult and G. Almuneau are with LAAS-CNRS, 7 av. du Colonel Roche, 31400 Toulouse, France (3) C. Aicardi is with CNES, 18 av. E. Belin, 31400 Toulouse, France (4) S. Parola is with IES, Université de Montpellier, CNRS, 860 Rue de St -Priest, 34095, Montpellier,

TABLE I .

 I INGAASN GROWTH CONDITIONS Degradation study of InGaAsN PIN solar cell under 1 MeV electron irradiation M. Levillayer 1,2,3 , S. Duzellier 1 , T. Nuns 1 , C. Inguimbert 1 , T. Le Cocq 1 , R. Rey 1 , C. Pons 1 , F. Olivié 2 , I. Massiot 2 , A. Arnoult 2 , C. Aicardi 3 , S. Parola 4 , G. Almuneau 2 , L. Artola 1

	Wafer	Structure	Surfactant	Growth Temperature	As/III
	A		Bi	465°C	12
	B		Ø	465°C	12
		p-i-n cells
	C		Ø	445°C	10
	D		Ø	485°C	10
	E	InGaAsN	Ø	465°C	11
	F	monolayer	Ø	465°C	8

ACKNOWLEDGMENT

We acknowledge the technical support from the LAAS-CNRS micro and nanotechnologies platform, a member of the French RENATECH network and the CNES for its financial support through R-S19/MT-9999-245 project. The authors also acknowledge F. Pichot from CTM University of Montpellier for the AuGeNiAu metallization, and Nicalos Vaissière and Jean Decobert from III-V Lab for the electrochemical voltagecapacitance measurements . This work was partly supported by the French program on « Investments for the future » managed by the National Agency for Research under contracts ANR-10-LABX-22-01-SOLSTICE.