Pierre Santucci

Sadia Diomandé

Isabelle Poncin

Laetitia Alibaud

Albertus Viljoen

Laurent Kremer

Chantal De Chastellier
email: chantal.dechastellier@wanadoo.fr

Stéphane Canaan
email: canaan@imm.cnrs.fr

Delineating the physiological roles of the PE and catalytic domain of LipY in lipid consumption in mycobacteria-infected foamy macrophages

Keywords: Electron microscopy, lipolysis, lipid bodies, intracytosolic lipid inclusions, Mycobacterium tuberculosis, M. bovis BCG

Within tuberculous granulomas, a sub-population of Mycobacterium tuberculosis resides inside foamy macrophages (FM) that contain abundant cytoplasmic lipid bodies (LB) filled with triacylglycerol (TAG). Upon fusion of LB with M. tuberculosiscontaining phagosomes, TAG is hydrolyzed and reprocessed by the bacteria into their own lipids, which accumulate as intracytosolic lipid inclusions (ILI). This phenomenon is driven by many mycobacterial lipases, among which LipY participates in the hydrolysis of host and bacterial TAG. However, the functional contribution of LipY's PE domain in TAG hydrolysis remains unclear. Herein, enzymatic studies were performed to compare the lipolytic activity of recombinant LipY and its truncated variant lacking the N-terminal PE domain, LipY(∆PE). Complementarily, an FM model was used where BMDM were infected with M. bovis BCG strains either overexpressing LipY or LipY(ΔPE) or carrying a lipY-deletion mutation prior to being exposed to TAG-rich VLDL. Results indicate that truncation of the PE domain correlates with increased TAG hydrolase activity. Quantitative electron microscopy analyses showed that (i) in the presence of lipase inhibitors, large ILI were not formed either because of an absence of LB due to inhibition of VLDL-TAG hydrolysis or inhibition of LB-neutral lipid hydrolysis by mycobacterial lipases; (ii) large ILI +3 in the strain overexpressing LipY(ΔPE) were reduced and, (iii) the number of ILI +3 profiles in the ΔlipY mutant was reduced by 50%. Overall, these results delineate the role of LipY and its PE domain in host and mycobacterial lipid consumption and show that additional mycobacterial lipases take part in these processes.

INTRODUCTION

With more than 10 million new cases and 1.7 million deaths in 2016, tuberculosis (TB), caused by Mycobacterium tuberculosis, remains one of the most devastating diseases worldwide [START_REF] Who | Global Tuberculosis Report[END_REF]. This serious illness is not fully treatable with current medication.

Prognosis of the disease depends on the host's ability to contain the bacilli at the site of infection within granulomas. Tuberculous granulomas are complex and dynamic immunological structures, which generate a wide range of microenvironments [START_REF] Irwin | Presence of multiple lesion types with vastly different microenvironments in C3HeB/FeJ mice following aerosol infection with Mycobacterium tuberculosis[END_REF][START_REF] Lenaerts | Heterogeneity in tuberculosis pathology, microenvironments and therapeutic responses[END_REF] that are assumed to drastically affect the tubercle bacilli physiological properties [START_REF] Lenaerts | Heterogeneity in tuberculosis pathology, microenvironments and therapeutic responses[END_REF][START_REF] Manina | Stress and host immunity amplify Mycobacterium tuberculosis phenotypic heterogeneity and induce nongrowing metabolically active forms[END_REF]. Inside the high diversity of TB pulmonary lesions found during active disease or latent infection, a distinct bacterial sub-population appears associated with a specific cell type consisting of foamy macrophages (FM) characterized by the presence of numerous triacylglycerol (TAG)-filled lipid bodies (LB) in their cytosol [START_REF] Irwin | Presence of multiple lesion types with vastly different microenvironments in C3HeB/FeJ mice following aerosol infection with Mycobacterium tuberculosis[END_REF][START_REF] Peyron | Foamy macrophages from tuberculous patients' granulomas constitute a nutrient-rich reservoir for M. tuberculosis persistence[END_REF][START_REF] Hunter | Pathology of postprimary tuberculosis in humans and mice: contradiction of long-held beliefs[END_REF]. To date, the exact role of FM in TB pathogenesis remains elusive, however these specialized lipid-rich cells have been described in both experimentally-infected animals and patients suggesting that they might play an essential role in granuloma formation and maturation processes. Several independent studies have demonstrated that pathogenic mycobacteria during in-vitro FM infection, are able to survive in a slow or non-replicating state [START_REF] Peyron | Foamy macrophages from tuberculous patients' granulomas constitute a nutrient-rich reservoir for M. tuberculosis persistence[END_REF][START_REF] Caire-Brandli | Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis[END_REF]. These experimental observations prompted the authors to propose that FM may also promote M. tb intracellular survival for extensive periods in vivo, although this remains to be demonstrated. A better understanding of how mycobacteria interact with and persist within FM is important to decipher this complex host-pathogen crosstalk that may sustain latent TB and, eventually, to identify new pharmacological approaches to control both active and latent TB. It is well-known that storage of large amounts of neutral lipids in the form of intracytosolic lipid inclusions (ILI) is one of the main characteristics of persistent mycobacteria [START_REF] Garton | Cytological and transcript analyses reveal fat and lazy persister-like bacilli in tuberculous sputum[END_REF]. ILI are mainly composed of triacylglycerols (TAG) resynthesized by mycobacterial TAG synthases from free fatty acids coming from the hydrolysis of host TAG [START_REF] Daniel | An acyl-CoA synthetase in Mycobacterium tuberculosis involved in triacylglycerol accumulation during dormancy[END_REF][START_REF] Daniel | Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages[END_REF][START_REF] Kapoor | Human granuloma in vitro model, for TB dormancy and resuscitation[END_REF][START_REF] Viljoen | MAB_3551c encodes the primary triacylglycerol synthase involved in lipid accumulation in Mycobacterium abscessus[END_REF]. Host lipid transfer to the phagosomal lumen occurs via mycobacteriuminduced fusion between LB and phagosomes [START_REF] Caire-Brandli | Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis[END_REF]. Lipids within ILI seem to serve as a source of carbon and energy prior to reactivation of dormant bacilli [START_REF] Kapoor | Human granuloma in vitro model, for TB dormancy and resuscitation[END_REF][START_REF] Dhouib | Watching intracellular lipolysis in mycobacteria using time lapse fluorescence microscopy[END_REF].

Although the origin of the ILI-containing lipids has become the focus of intense research [START_REF] Peyron | Foamy macrophages from tuberculous patients' granulomas constitute a nutrient-rich reservoir for M. tuberculosis persistence[END_REF][START_REF] Daniel | Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages[END_REF][START_REF] Kapoor | Human granuloma in vitro model, for TB dormancy and resuscitation[END_REF][START_REF] Santucci | Experimental Models of Foamy Macrophages and Approaches for Dissecting the Mechanisms of Lipid Accumulation and Consumption during Dormancy and Reactivation of Tuberculosis[END_REF][START_REF] Daniel | The perilipin-like PPE15 protein in Mycobacterium tuberculosis is required for triacylglycerol accumulation under dormancy-inducing conditions[END_REF], the catalytic steps involved in the formation of mycobacterial ILI are not completely understood [START_REF] Dedieu | Mycobacterial lipolytic enzymes: a gold mine for tuberculosis research[END_REF][START_REF] Singh | Lipid hydrolizing enzymes in virulence: Mycobacterium tuberculosis as a model system[END_REF]. It is obvious, however, that hydrolysis of host TAG released into the phagosome involves specific mycobacterial lipolytic enzymes, either secreted by the bacilli into the phagosomal lumen or associated with the outermost layer of the mycobacterial cell wall [START_REF] Dedieu | Mycobacterial lipolytic enzymes: a gold mine for tuberculosis research[END_REF][START_REF] Daleke | Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) protein domains target LipY lipases of pathogenic mycobacteria to the cell surface via the ESX-5 pathway[END_REF][START_REF] Dhouib | A monoacylglycerol lipase from Mycobacterium smegmatis Involved in bacterial cell interaction[END_REF][START_REF] Schué | Two cutinase-like proteins secreted by Mycobacterium tuberculosis show very different lipolytic activities reflecting their physiological function[END_REF] whereas lipolytic enzymes involved in hydrolysis of ILI-contained TAG must be located in the mycobacterial cytosol [START_REF] Kapoor | Human granuloma in vitro model, for TB dormancy and resuscitation[END_REF][START_REF] Deb | A novel lipase belonging to the hormone-sensitive lipase family induced under starvation to utilize stored triacylglycerol in Mycobacterium tuberculosis[END_REF][START_REF] Mishra | Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY[END_REF].

Among the many mycobacterial lipases found in M. tuberculosis, LipY (Rv3097c) appears as a major player in the degradation of TAG within ILI under in vitro growth conditions mimicking dormancy [START_REF] Kapoor | Human granuloma in vitro model, for TB dormancy and resuscitation[END_REF][START_REF] Deb | A novel lipase belonging to the hormone-sensitive lipase family induced under starvation to utilize stored triacylglycerol in Mycobacterium tuberculosis[END_REF][START_REF] Mishra | Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY[END_REF]. LipY belongs to the hormone-sensitive lipase family and displays an N-terminal PE domain that is specific to pathogenic mycobacterial species [START_REF] Deb | A novel lipase belonging to the hormone-sensitive lipase family induced under starvation to utilize stored triacylglycerol in Mycobacterium tuberculosis[END_REF][START_REF] Mishra | Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY[END_REF][START_REF] Delorme | MmPPOX inhibits Mycobacterium tuberculosis lipolytic enzymes belonging to the hormone-sensitive lipase family and alters mycobacterial growth[END_REF]. Relevant studies demonstrated that PE proteins are exported to the cell wall via the type VII secretion system ESX-5 after recognition of the specific amino-acid sequence YxxxD/E contained in the N-terminal PE domain [START_REF] Daleke | Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) protein domains target LipY lipases of pathogenic mycobacteria to the cell surface via the ESX-5 pathway[END_REF][START_REF] Abdallah | Type VII secretion--mycobacteria show the way[END_REF][START_REF] Abdallah | PPE and PE_PGRS proteins of Mycobacterium marinum are transported via the type VII secretion system ESX-5[END_REF][START_REF] Daleke | General secretion signal for the mycobacterial type VII secretion pathway[END_REF]. Among the enzymes of the PE family, only two members, PE-PGRS11 (or Rv0754), a functional phosphoglycerate mutase [START_REF] Chaturvedi | The multifunctional PE_PGRS11 protein from Mycobacterium tuberculosis plays a role in regulating resistance to oxidative stress[END_REF] and LipY, have been biochemically characterized. LipY was proposed to be a major contributor to the breakdown of stored TAG [START_REF] Deb | A novel lipase belonging to the hormone-sensitive lipase family induced under starvation to utilize stored triacylglycerol in Mycobacterium tuberculosis[END_REF][START_REF] Mishra | Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY[END_REF]. In addition, a recombinant Mycobacterium smegmatis strain overexpressing the full-length LipY, exhibited a reduced TAG hydrolytic activity as compared to a LipY variant shortened by its N-terminal PE domain, LipY(ΔPE), suggesting that the PE domain may negatively modulate the activity of LipY [START_REF] Mishra | Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY[END_REF][START_REF] Garrett | Modulation of the Activity of Mycobacterium tuberculosis LipY by Its PE Domain[END_REF].

In the present study, the experimental model system of VLDL-driven FM [START_REF] Caire-Brandli | Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis[END_REF] was used to delineate the physiological role of the PE domain of LipY in the hydrolysis of both host-derived TAG and mycobacterial TAG within ILI. Reasons for choosing this model system have been described in detail elsewhere [START_REF] Caire-Brandli | Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis[END_REF][START_REF] Viljoen | MAB_3551c encodes the primary triacylglycerol synthase involved in lipid accumulation in Mycobacterium abscessus[END_REF][START_REF] Santucci | Experimental Models of Foamy Macrophages and Approaches for Dissecting the Mechanisms of Lipid Accumulation and Consumption during Dormancy and Reactivation of Tuberculosis[END_REF][START_REF] Neyrolles | Mycobacteria and the greasy macrophage: getting fat and frustrated[END_REF]. After internalization of VLDL by receptor-mediated endocytosis, the neutral lipids of this lipoprotein will undergo hydrolysis in lysosomes. This will provide fatty acids for the subsequent biosynthesis of neutral lipids, including TAG, between the two leaflets of the endoplasmic reticulum, where LB, consisting of a phospholipid monolayer encapsulating neutral lipids, bud of. This model system generates defined conditions for inducing the formation or removal of LB, which triggers ILI formation or consumption, and helps delineating the role of host cell or mycobacterial lipases in these processes.

We first show that addition of lipase inhibitors, during exposure of infected cells to VLDL, inhibits the accumulation of lipids in the form of ILI by affecting either the macrophage lysosomal TAG hydrolase and, hence, LB formation, or mycobacterial lipases involved in the hydrolysis of host TAG delivered to phagosomes. Next, we demonstrate the modulatory role of its N-terminal PE domain on LipY function by, as a first approach, comparing the catalytic activity of purified LipY and LipY(ΔPE) variants using a range of lipids with various chain lengths. As a second approach, we used quantitative electron microscopy (EM) methods to examine differences in mycobacterial ILI formation in bone marrow-derived mouse macrophages (BMDM) exposed to VLDL to become foamy after they were infected with M. bovis BCG strains overexpressing different LipY variants or in which the lipY gene was deleted. This study indicates that other lipases, in addition to LipY, are likely to be involved in ILI formation. Removal of VLDL normally leads to the loss of TAG-containing ILI [START_REF] Caire-Brandli | Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis[END_REF]. When lipase inhibitors were added after exposure to VLDL no loss of ILI occurred, a result that emphasizes the participation of several lipolytic enzymes in the hydrolysis of bacterial cytosolic TAG. The M. bovis BCG Pasteur 1173P2 strain used for overexpression experiments was grown at 37°C with shaking (160 rpm) in Middlebrook 7H9 medium supplemented with 0.05% Tween 80 (v/v), 0.2% glycerol (v/v) and 10% Albumin Dextrose Catalase (v/v) (ADC). Preparation of M. smegmatis and M. bovis BCG electrocompetent cells and electroporation procedures were performed as described previously [START_REF] Goude | Electroporation of mycobacteria[END_REF]. Transformants were selected on Middlebrook 7H11 agar supplemented with 10% ADC and either 50 µg/mL hygromycin or 50 µg/mL kanamycin. Plates were incubated at 37°C for 3-5 days for M. smegmatis and for three weeks for M. bovis BCG.

MATERIALS AND METHODS

Reagents

Complementation of M. bovis BCG ΔlipY strain.

A M. bovis BCG mutant lacking the lipY gene (ΔlipY) was grown in the presence of 50 µg/mL hygromycin [START_REF] Low | Triacylglycerol utilization is required for regrowth of in vitro hypoxic nonreplicating Mycobacterium bovis bacillus Calmette-Guerin[END_REF].

Complemention was performed by introducing pMV261::lipY [START_REF] Mishra | Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY[END_REF] prior to selection on 7H11 Middlebrook agar medium supplemented with 10% ADC and kanamycin. Presence of the plasmid was checked by PCR and expression of LipY confirmed by immunoblotting.

Briefly, bacterial lysates from the WT, ΔlipY and complemented strain (ΔlipY::Comp) were normalized for total protein content, electrophoretically separated on a 12% SDS-PAGE gel and transferred onto a nitrocellulose membrane using a Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were then saturated with 1% bovine serum albumin (BSA) in PBS, 0.1% Tween 20 and probed for 1 hour with mouse antiserum raised against LipY at a 1/2,500 dilution. After extensive washing, the membrane was incubated with horseradish peroxidase-conjugated IgG anti-mouse antibodies (Sigma-Aldrich). The GroEL2 protein was used as protein loading control and was revealed using the HisProbe TM HRP conjugate (Thermo-Scientific) which recognizes the natural poly-Histidine sequence present in its C-terminal domain [START_REF] Kong | Mycobacterium tuberculosis expresses two chaperonin-60 homologs[END_REF][START_REF] Ojha | GroEL1: a dedicated chaperone involved in mycolic acid biosynthesis during biofilm formation in mycobacteria[END_REF]. Detection was achieved using the Pierce TM ECL Western Blotting substrate solution (Thermo-Scientific) and visualized using the ChemiDoc TM MP Imaging System (Bio-Rad).

Cloning, expression and purification of LipY and LipY(ΔPE). The lipY and lipY(ΔPE)

genes were amplified by PCR using M. tuberculosis H37Rv genomic DNA and cloned into pSD26 under the control of the acetamide inducible promoter and carrying a hygromycin resistance cassette [START_REF] Daugelat | The RD1 proteins of Mycobacterium tuberculosis: expression in Mycobacterium smegmatis and biochemical characterization[END_REF] or into pMV261 (36) downstream of the hsp60 promoter and containing a kanamycin resistance cassette, as reported previously [START_REF] Mishra | Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY[END_REF].

DNA sequence analysis of each insert was performed by GATC Biotech (Germany).

Expression and purification of recombinant LipY or LipY(ΔPE) were performed as previously reported [START_REF] Brust | Mycobacterium tuberculosis lipolytic enzymes as potential biomarkers for the diagnosis of active tuberculosis[END_REF] with some minor modifications. Briefly, M. smegmatis mc 2 155 groEL1ΔC strains carrying pSD26-lipY and pSD26-lipY(ΔPE), were used to inoculate 10 mL of 7H9 medium containing 50 μg/mL hygromycin. After three days of incubation at 37°C with shaking, 10 mL of the preparation were used to inoculate 400 mL of culture medium for a large-scale production. Cultures were grown at 37°C with shaking (220 rpm) until an OD600nm value between 2.5 and 3 was reached. Expression of recombinant proteins was induced by adding acetamide to a final concentration of 0.2% (w/v) for 16 h. Bacteria were harvested, resuspended in ice-cold buffer A (10 mM Tris/HCl pH 8.0, 150 mM NaCl) (30 mL) containing 1% N-lauroylsarcosine and lysed using a French Press set 1100 psi. After centrifugation, the supernatant (S1) was recovered while the resulting pellet was resuspended in buffer A (30 mL) and sonicated twice for 30 s with 30 s breaks between each cycle and stirred overnight at 4°C. After centrifugation, the new supernatant (S2) was pooled with S1 supernatant and the mixture was loaded onto a Ni 2+ -NTA resin equilibrated with buffer A. The column was subsequently washed with buffer A without detergent prior to elution with increasing concentrations of imidazole. The eluted fractions were analysed by performing 12% SDS-PAGE as described previously [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF].

Fractions containing pure proteins were pooled, dialysed overnight against buffer A and concentrated by ultrafiltration to a final concentration of 0.6 mg/mL and stored at -80°C.

Theoretical physical properties (molecular mass, extinction coefficient at 280 nm and isoelectric point) of both proteins containing the His6-tag at the C-Terminal position were obtained from the ProtParam tool (http://ca.expasy.org/tools/protparam.html).

Enzyme activity measurements using the pH-stat technique. Enzymatic hydrolysis of emulsions of mono-, di-and triacylglycerol namely monobutyrin (MC4), monolein (MC18), diolein (DC18), tributyrin (TC4) and trioctanoin (TC8) were monitored titrimetrically for at least 5 min at 37°C using a pH-stat (Metrohm 718 STAT Titrino; Metrohm Ltd., Herisau, Switzerland). Assays were performed in 2.5 mM Tris-HCl buffer (pH 7.5) containing 300 mM NaCl and 3 mM NaTDC [START_REF] Delorme | MmPPOX inhibits Mycobacterium tuberculosis lipolytic enzymes belonging to the hormone-sensitive lipase family and alters mycobacterial growth[END_REF][START_REF] Point | Synthesis and kinetic evaluation of cyclophostin and cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases[END_REF]. Free fatty acids released were automatically titrated with 0.1 M NaOH to maintain a fixed end-point pH value of 7.5. The specific activity of both enzymes was expressed in units per mg of pure enzyme. One unit corresponds to the release of 1 µmole of fatty acid per minute.

Lipase activity assays on TAG from Pomegranate oil. Corning UV 96-well microplates were coated as recently described (40) using TAG from Pomegranate oil, containing up to 80% punicic acid (C18:3) equally present at the 3 positions of the glycerol backbone.

The lipase activity was measured at 37°C in 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl, 6 mM CaCl2, 1 mM EDTA, 0.001% (w/v) 3,5-di-tert-4-butylhydroxytoluene (BHT) and 3 mg/mL β-Cyclodextrine (β-CD). The formation of the β-CD/free punicic acid complex was continuously monitored at 275 nm for 60 min.

In silico Protein Modeling. Three-dimensional structural models of LipY and LipY(ΔPE) proteins were generated with the automatic protein structure homology modeling server using the I-Tasser software program [START_REF] Roy | I-TASSER: a unified platform for automated protein structure and function prediction[END_REF][START_REF] Zhang | I-TASSER server for protein 3D structure prediction[END_REF]. The alignment of the LipY and LipYΔPE sequences were performed using Multalin multiple sequence alignment [START_REF] Corpet | Multiple sequence alignment with hierarchical clustering[END_REF] and the result was displayed with ESPript [START_REF] Robert | Deciphering key features in protein structures with the new ENDscript server[END_REF]. The structural overlay and figures were drawn using the PyMOL Molecular Graphics System (version 1.8.6.0, Schrödinger, LLC).

Infection of bone marrow-derived mouse macrophages (BMDM) with recombinant

M. bovis BCG strains. Bone marrow cells were isolated from the femurs of 6-to 8-week-old C57BL/6 female mice and seeded onto tissue culture dishes (Falcon; Becton Dickinson Labware, Meylan, France) 35 mm in diameter (4x10 5 cells per dish). The culture medium was DMEM with high glucose (1 g liter -1 ) and high carbonate (3.7 g liter - 1 ) concentrations supplemented with 10% heat-inactivated FBS, 10% L-cell conditioned medium (a source of colony-stimulating factor 1, CSF-1), and 2 mM L-glutamine. Five days after seeding, the adherent cells were washed twice with DMEM and refed with complete medium. Medium was then renewed on day 6. No antibiotics were added. On day 7, the cells were infected for 4 h at 37°C with wild type or different recombinant M.

bovis BCG strains at a multiplicity of infection (MOI) of 5 for EM studies, washed in 4 changes of ice-cold PBS to eliminate non-ingested bacteria, and further incubated in complete medium devoid of antibiotics. For long-term cultures, the medium was changed twice a week.

Treatment with VLDL and chase after treatment. After active replication of M. bovis BCG for 6 days, infected macrophages were exposed to Very Low Density Lipoprotein (VLDL), for 24 h. The volume of VLDL was adjusted so as to expose cells (1 x 10 6 per dish) to 180 µg TAG per mL of medium. In some instances, infected cells were exposed to VLDL simultaneously with either tetrahydrolipstatin (THL also named Orlistat, 60 µg/mL in ethanol) or MmPPOX (15 µg/mL in ethanol) for 24 h [START_REF] Delorme | MmPPOX inhibits Mycobacterium tuberculosis lipolytic enzymes belonging to the hormone-sensitive lipase family and alters mycobacterial growth[END_REF]. Cells were also exposed to VLDL for 24 h, washed and incubated in a VLDL-free fresh medium with or without THL or MmPPOX for 24 h.

Processing for conventional electron microscopy (EM).

Cells were fixed for 1 h at room temperature with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) containing 0.1 M sucrose, 5 mM CaCl2, and 5 mM MgCl2, washed with complete cacodylate buffer, and post-fixed for 1 h at room temperature with 1% osmium tetroxide in the same buffer devoid of sucrose. They were washed with buffer, scraped off the dishes, concentrated in 2% agar in cacodylate buffer, and treated for 1 h at room temperature with 1% uranyl acetate in 30% methanol. Samples were dehydrated in a graded series of ethanol solutions and embedded in Spurr resin. Thin sections (70 nm thick) were stained with 1% uranyl acetate in distilled water and then with lead citrate.

Quantification and statistical analysis. At the time points indicated in the figures, 150 to 300 intraphagosomal mycobacteria per sample were examined under an EM to score the percentage of each category of M. bovis BCG ILI profiles. The cells were examined at random, and care was taken to avoid serial sections. Histograms represent the mean ± standard deviation of at least three independent counts. Statistical analyses were performed using GraphPad Prism 4.03 and differences were considered statistically significant when P-values were ≤ 0.05 using two tailed Student's t-tests.

RESULTS

ILI formation depends on lipid processing by macrophage and mycobacterial

lipases. Previously, we have shown the dependence of ILI formation on the presence of LB in the host, confirmed the finding that host TAG provided the bulk of lipid for ILI formation and demonstrated that VLDL-derived TAG must first be processed by host lysosomal lipases [START_REF] Caire-Brandli | Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis[END_REF]. The requirement of host lipases for the release of fatty acids had been tested by exposing M. avium-infected macrophages for 24 h to VLDL in the presence of the anti-obesity drug, tetrahydrolipstatin (THL or Orlistat ® ). THL inhibits human digestive lipases within the gastro-intestinal tract [START_REF] Hadvàry | Inhibition of pancreatic lipase in vitro by the covalent inhibitor tetrahydrolipstatin[END_REF] as well as a variety of other serine-hydrolases [START_REF] Yang | Activity-based proteome profiling of potential cellular targets of Orlistat--an FDA-approved drug with anti-tumor activities[END_REF][START_REF] Nelson | The use of orlistat in the treatment of obesity, dyslipidaemia and Type 2 diabetes[END_REF], including mycobacterial LipY, a major enzyme responsible for hydrolysis of ILI-contained TAG [START_REF] Deb | A novel lipase belonging to the hormone-sensitive lipase family induced under starvation to utilize stored triacylglycerol in Mycobacterium tuberculosis[END_REF].

In the present work, we applied the above procedure to cells infected with wildtype M. bovis BCG. As described in our previous study [START_REF] Caire-Brandli | Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis[END_REF], endocytosis of VLDL and transfer to lysosomes was not affected by THL but TAG and derivatives in VLDL were not degraded as usual. These lipids were easily recognized by their electron-transparency and were seen to fill the entire lumen of most lysosomes, leaving only small spaces with the usual electron-dense lysosomal contents (Fig. 1A). This was not observed when infected cells were exposed to VLDL only, in which case lysosomes keep their normal electron-dense appearance (Fig. 1B). In the presence of THL, very few, if any, LB were observed with an average value of approximately 0.32 LB per cell (Supplemental Material, Fig. S1). As expected, with this extremely low level of LB (approximately 23 times less than the untreated cells), mycobacterial profiles with large ILI filling most of the mycobacterial cytoplasm (defined as ILI +3 in (7)) were infrequent (Fig. 1C) as opposed to when LB were formed in the presence of VLDL only (Fig 1D ). A quantitative evaluation (Fig. 1G) showed that the amount of ILI +3 and ILI +2 profiles remained low, less than 1% and 23% ±1.1, respectively, in presence of THL vs 25% ± 7.4 and 45% ± 2, respectively, in the absence of this lipase inhibitor. Consequently, the amount of ILI (+1/-) profiles was 2.5 fold higher in presence of THL (with: 78% ± 9.6; without: 31% ± 0.5).

These results suggest that mycobacterial lipases were unable to process TAG in the VLDL core directly and that VLDL-derived TAG first had to be processed by lysosomal lipases in order to hydrolyze TAG into diacylglycerides (DAG) and monoacylglycerides (MAG) and FFA for subsequent re-processing into TAG and accumulation in LB. Because no LB were formed, it was not possible to gain information on the role of mycobacterial lipases on host TAG hydrolysis. However, THL has also been reported as a non-specific inhibitor of a wide range of serine-hydrolases from both mammals and bacterial species [START_REF] Dhouib | Watching intracellular lipolysis in mycobacteria using time lapse fluorescence microscopy[END_REF][START_REF] Dhouib | A monoacylglycerol lipase from Mycobacterium smegmatis Involved in bacterial cell interaction[END_REF][START_REF] Hadvàry | The lipase inhibitor tetrahydrolipstatin binds covalently to the putative active site serine of pancreatic lipase[END_REF][START_REF] Ravindran | Targeting Lipid Esterases in Mycobacteria Grown Under Different Physiological Conditions Using Activity-based Profiling with Tetrahydrolipstatin (THL)[END_REF] and, therefore one cannot exclude that mycobacterial enzymes are also inhibited by THL, thereby impacting ILI formation.

We then resorted to the oxadiazolone inhibitor, MmPPOX, known to inhibit a variety of mycobacterial serine-hydrolases including those of the hormone-sensitive lipase (HSL) family [START_REF] Ali | Use of an inhibitor to identify members of the hormone-sensitive lipase family[END_REF], and more specifically LipY [START_REF] Daleke | Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) protein domains target LipY lipases of pathogenic mycobacteria to the cell surface via the ESX-5 pathway[END_REF][START_REF] Delorme | MmPPOX inhibits Mycobacterium tuberculosis lipolytic enzymes belonging to the hormone-sensitive lipase family and alters mycobacterial growth[END_REF]. After exposure of M. bovis BCG-infected cells to VLDL in the presence of MmPPOX, host lysosomes were not affected in the same way as during THL treatment. Here, most lysosomes retained their usual electron-dense appearance. Less than 25% of the lysosomes contained small lipid droplets (Fig. 1E) and only 10% were filled with TAG and derivatives as in the case of macrophages exposed to VLDL with THL. Notably, with MmPPOX the relative number of LB per cell was 6 to 7 times higher than with a THL treatment (Supplemental Material, Fig. S1), with an average value of 2.1 LB per cell (thus leading to a 3.5-fold reduction in LB content, in comparison with an untreated sample). These data suggest that host lysosomal lipases were poorly affected by MmPPOX. As a result, VLDL-derived TAG could be broken down into di-and mono-glycerides and fatty acids for re-processing into TAG and accumulation in LB.

Strikingly, during MmPPOX treatment mycobacterial profiles with large ILI were not observed as demonstrated (Fig. 1F). A quantitative evaluation of mycobacterial profiles containing the different categories of ILI showed that those with large (ILI +3 ) and medium-sized (ILI +2 ) ILI reached less than 5% and 20%, respectively, of the amount generated by cell exposure to VLDL in absence of MmPPOX (Fig. 1G). These data suggest that the cell-surface exposed mycobacterial lipases involved in hydrolysis of host TAG delivered to wild-type M. bovis BCG-containing phagosomes are involved in ILI formation and that MmPPOX appears to inhibit the mycobacterial lipases more efficiently than THL. By comparing the LB/ILI relative ratio from the two treated samples, a clear difference in the effects of the two inhibitors was observed where LB/ILI +3 and LB/ILI +2 ratios were 9.8 and 12.4 times higher, respectively, in MmPPOX than in THL treated samples (Supplemental Material, Fig. S1).

Altogether, these data further support that in our system, VLDL has to be processed by host lipases in order to newly synthesize host LB, and that these neutral lipid-rich organelles have to be delivered within mycobacteria-containing phagosomes for subsequent hydrolysis into free fatty acids, which will allow ILI formation. However, these experiments do not allow to identify specific mycobacterial lipase(s) involved in this process, although the LipY form associated with the mycobacterial surface remains the most likely candidate.

Biochemical characterization of rLipY and rLipY(ΔPE). The LipY hydrolase from

M. tuberculosis has a dual location, firstly in the mycobacterial cytosol where it has direct access to ILI for subsequent lipid hydrolysis [START_REF] Deb | A novel lipase belonging to the hormone-sensitive lipase family induced under starvation to utilize stored triacylglycerol in Mycobacterium tuberculosis[END_REF][START_REF] Mishra | Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY[END_REF] and, second, at the mycobacterial cell surface in a truncated/mature form following removal of its N-terminal PE domain [START_REF] Daleke | Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) protein domains target LipY lipases of pathogenic mycobacteria to the cell surface via the ESX-5 pathway[END_REF][START_REF] Deb | A novel lipase belonging to the hormone-sensitive lipase family induced under starvation to utilize stored triacylglycerol in Mycobacterium tuberculosis[END_REF][START_REF] Mishra | Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY[END_REF][START_REF] Fishbein | Phylogeny to function: PE/PPE protein evolution and impact on Mycobacterium tuberculosis pathogenicity[END_REF]. This dual localization has been explained by Daleke et al. (18) who demonstrated that the PE domain of LipY is essential for secretion by ESX-5 [START_REF] Daleke | Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) protein domains target LipY lipases of pathogenic mycobacteria to the cell surface via the ESX-5 pathway[END_REF][START_REF] Abdallah | PPE and PE_PGRS proteins of Mycobacterium marinum are transported via the type VII secretion system ESX-5[END_REF][START_REF] Daleke | General secretion signal for the mycobacterial type VII secretion pathway[END_REF].

It is not known, however, whether this secretion also requires additional partners such as PPE proteins, which are known to form pairs with PE proteins. Mishra et al. proposed that the PE domain not only plays a role in the recognition by ESX-5 for secretion of LipY but also contributes to the enzymatic activity of the protein [START_REF] Mishra | Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY[END_REF]. Overexpression of LipY(ΔPE), a LipY version lacking its N-terminal PE domain, in M. smegmatis was associated with increased activity as compared to the strain overexpressing the full-length protein [START_REF] Mishra | Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY[END_REF]. Nevertheless, whether lack of the PE domain directly affects LipY activity or whether the increased activity of LipY(ΔPE) results from an indirect effect, presumably due to the absence of a possible partner, remains unknown. To address this issue, we compared the enzymatic activity of purified LipY and LipY(ΔPE) recombinant proteins.

Because the E. coli expression system failed to express large amounts of LipY in an active form, we opted for M. smegmatis mc 2 155 groEL1ΔC as a surrogate host which allowed producing around 30 mg of pure and active recombinant LipY from a 400 mL culture volume (Fig. 2A). Regarding rLipY(ΔPE), 9 mg of pure (Fig. 2A) and active enzyme were obtained from equivalent cultures, thus indicating that the PE domain is not needed for proper folding of the protein. This was confirmed by further biochemical analyses and circular dichroism spectra determination of both recombinant enzymes (data not shown). The lower rLipY(ΔPE) yield could rely on the absence of the PE domain that exposes the lipid binding site of LipY, promoting a higher degree of interactions between molecules and excessive protein aggregation.

The specific activity of rLipY and rLipY(ΔPE) was determined using a range of lipid substrates differing in their chain lengths and by combining titrimetric and spectrophotometric techniques. Regardless of the lipid substrates used, rLipY(ΔPE) exhibited higher specific activities (1.2 to 2.5 times depending on the substrate) than rLipY (Table 1). Both proteins were preferentially active on short-chain emulsified MAG and TAG, i.e., monobutyrin (61.0 ± 2.0 and 92.0 ± 5.0 U/mg, respectively) and tributyrin (129.0 ± 6.0 and 267.0 ± 24.0 U/mg, respectively) and displayed the same chain length specificity since their respective specific activities decreased gradually as the substrate chain length increased (Table 1). These results unambiguously show that the lipase activity of LipY is significantly enhanced in the absence of its PE domain, consistent with previous findings [START_REF] Mishra | Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY[END_REF]. Moreover, LipY has a nonspecific lipase activity able to hydrolyse DAG and MAG as well as TAG. Therefore, LipY has the potential to degrade total host TAG converting them into glycerol and free fatty acids, products that can subsequently be directly absorbed by the bacteria.

The N-terminus contains 4 α-helices (α1 to α4), all lacking in LipY(ΔPE) (Fig. 2B).

Molecular modelling was performed allowing us to propose the overall structures of both the full-length and the truncated LipY proteins (Fig. 2C) and to address whether these secondary structures may alter either the optimal recognition and/or binding of the substrates to LipY. The α1-α4 helices of the PE domain are connected to a linker unit composed of three helices (α5-α7), presumably providing a high degree of flexibility to the PE and core (C-terminal) domains. The latter, comprising the active site of LipY belongs to the α/β hydrolase fold family and is composed of a central β-sheet (β1-β8 strands) surrounded by nine α-helices. Similar to most lipases, the catalytic serine (Ser309) is located in a nucleophile elbow between the β5 strand and the α13 helix. The presence of the PE domain (Fig. 2C) clearly reduces the accessibility of the active site to the substrate. Removal of the PE domain allows the linker portion (green) to move to the side of the catalytic domain, thus opening access to the active site. This large opening presumably allows a better accommodation of the lipid substrates into the active site, thereby contributing to the increased enzymatic activity of LipY(ΔPE).

Distribution of ILI profiles in M. bovis BCG strains over-expressing various LipY

variants in VLDL-driven FM. To investigate the functional contribution of the PE domain of LipY in host TAG hydrolysis and consequently ILI formation, macrophages were infected with various M. bovis BCG strains for 6 days, before being exposed to VLDL for 24 h to induce FM formation [START_REF] Caire-Brandli | Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis[END_REF]. The strains used in this experiment were the wild-type BCG strain (WT), which is characterized by the native expression of LipY and was used as reference, and three other recombinant BCG strains harboring distinct pMV261derived constructs, leading to the overproduction of either LipY, LipY(∆PE) or LipY(∆PE) in which Ser309 was replaced by an Ala residue (LipY(∆PE) S309A ) yielding a catalyticallyinactive protein [START_REF] Mishra | Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY[END_REF]. Cells were then fixed and processed for EM and profiles of intracellular mycobacteria were examined for the presence and extent of ILI formation in both VLDL-treated and untreated macrophages. As before [START_REF] Caire-Brandli | Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis[END_REF], intracellular mycobacteria were divided into 4 categories according to their ILI size (Figs. 3, A-D). ILI -profiles with no ILI (Fig. 3A), ILI +1 for profiles displaying a few small ILI 0.1 µm in width at most (Fig. 3B), ILI +2 for profiles displaying several ILI approximately 0.2 to 0.3 µm in width (Fig. 3C), and ILI +3 for profiles displaying several ILI approximately 0.4 to 0.5 µm in width and occupying most of the mycobacterial cytoplasm (Fig. 3D). The relative abundance of each type of ILI profile was scored on approximately 200 different mycobacterial profiles per sample (Fig. 3E). As expected, in the absence of exposure to VLDL, 95% of the M. bovis BCG profiles were ILI (+1/-) , regardless of the strain (data not shown). In contrast, after a 24 h exposure to VLDL, ILI +2 and ILI +3 were predominantly present among the different M. bovis BCG strains (Fig. 3E) as already found with M. avium [START_REF] Caire-Brandli | Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis[END_REF]. Further examination of the quantitative data showed that both the WT and the LipY-overexpressing strain displayed similar amounts of ILI profiles of each category with ILI +3 : 27% ± 4.8 and 25% ± 4.1; ILI +2 : 43% ± 3 and 40% ± 4.1; ILI (+1/-) : 29% ± 3.9 and 35% ± 6.8, respectively.

Likewise, and as expected, the relative abundance of the 4 types of ILI profiles in the strain over-expressing the catalytically-inactive form LipY(∆PE) S309A was similar to the ones scored in the WT strain and the strain overexpressing LipY (Fig. 3E). In sharp contrast with these strains, overexpression of LipY(∆PE) resulted in a substantial drop in the percentage of ILI +3 (15% ± 4.4 vs. 27% ± 4.8) and a concomitant increase in ILI (+1/-) profiles (50% ± 7.5 vs. 29% ± 3.9), as compared to the WT strain. Determination of the ILI 3+ /ILI +1/-ratio profiles showed that overexpression of LipY(∆PE) triggers important changes in the intracellular pool of ILI in comparison with the full-length LipY or the inactive form LipY(∆PE) S309A (Fig. 3E). These data are fully consistent with the increased in vitro activity of rLipY(∆PE) over rLipY (Table 1).

Additional mycobacterial lipases contribute to host TAG hydrolysis and ILI

formation. Despite the primary role of LipY in host lipid hydrolysis, it is very likely that additional mycobacterial lipases participate in this process, a hypothesis emphasized by the fact that the genome of M. tuberculosis encodes for at least 24 putative lipases [START_REF] Dhouib | A monoacylglycerol lipase from Mycobacterium smegmatis Involved in bacterial cell interaction[END_REF][START_REF] Schué | Two cutinase-like proteins secreted by Mycobacterium tuberculosis show very different lipolytic activities reflecting their physiological function[END_REF][START_REF] Delorme | MmPPOX inhibits Mycobacterium tuberculosis lipolytic enzymes belonging to the hormone-sensitive lipase family and alters mycobacterial growth[END_REF][START_REF] Low | Triacylglycerol utilization is required for regrowth of in vitro hypoxic nonreplicating Mycobacterium bovis bacillus Calmette-Guerin[END_REF][START_REF] West | Cutinase-like proteins of Mycobacterium tuberculosis: characterization of their variable enzymatic functions and active site identification[END_REF]. To test this hypothesis, macrophages were infected with a lipY-deleted M. bovis BCG mutant before being exposed to VLDL for 24 h. The cells were then fixed and processed for EM and profiles of intracellular mycobacteria were examined for the presence and extent of ILI formation in both the WT and mutant strains. The profiles of the mutant clearly displayed smaller ILI than those of the WT strain (Fig. 4A vs 4C). The relative abundance of each category of ILI was then scored on approximately 150 different mycobacterial profiles per sample (Fig. 4E). Although all categories of ILI were found in the profiles of the lipY mutant, the percentage of ILI +3 profiles was reduced by approximately 50% (16% ± 5.8 vs. 35% ± 2.9) with a concomitant increase in the percentage of ILI +1/-(46% ± 10.9 vs. 27% ± 5.5 ) with respect to the WT strain. Functional complementation of the ∆lipY mutant was performed with pMV261::lipY, which allows constitutive expression of the full-length lipY leading to the strain ΔlipY::Comp. Production of the protein was confirmed by immunoblotting using polyclonal antibody directed against the M. tuberculosis LipY protein (Fig. 4D). As mentioned earlier [START_REF] Daleke | Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) protein domains target LipY lipases of pathogenic mycobacteria to the cell surface via the ESX-5 pathway[END_REF][START_REF] Mishra | Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY[END_REF], LipY is not produced by wild-type M. bovis BCG in vitro under standard growth conditions explaining the lack of a reactive band in the corresponding crude lysate. In contrast, a specific immunoreactive band was detected in ΔlipY::Comp, indicating that LipY is constitutively produced in this strain. Thus, ΔlipY::Comp was used to infect macrophages for six days, before being exposed to VLDL for 24 h. Analysis of the ILI profile indicates that complementation restores the WT phenotype, characterized by 39% ± 5.3 and 23% ± 4.2 for ILI 3+ and ILI +1/-, respectively (Fig. 4B and4E). Likewise, the ILI 3+ /ILI +1/-ratio was severely impacted in ΔlipY and was restored upon functional complementation of the mutant, clearly confirming the impact of the lipY deletion on intrabacterial lipid accumulation (Fig 4F ).

Taken collectively, these results reveal the major contribution of LipY in the breakdown of host-derived TAG within the phagosomal lumen and highlight the role of additional mycobacterial lipases in hydrolysis of host TAG and accumulation of TAG in the form of large ILI.

Consumption of TAG within ILI correlates with the reduction of LB and the activity of cytosolic mycobacterial lipases.

To dissect the physiological link between LB in FM and ILI in mycobacteria, we had previously investigated whether the formation of large ILI (ILI +3 ) could be reversed by removal of VLDL [START_REF] Caire-Brandli | Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis[END_REF]. After exposure of infected macrophages to VLDL for 24 h, the infected cells had been washed and re-incubated in fresh medium without lipoprotein. Within 24 h after removal of VLDL, infected cells had lost their LB and ILI +3 were no longer visible in the mycobacterial profiles [START_REF] Caire-Brandli | Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis[END_REF], thus demonstrating the direct link between the presence/absence of LB and ILI formation/consumption. The same method applied to M. bovis BCG-infected cells and the present work yielded comparable results (Fig. 5D).

To determine whether mycobacterial lipases were involved in ILI consumption, we applied the same chase strategy, but in the absence or presence of lipase inhibitors. After exposure of WT M. bovis BCG-infected cells to VLDL for 24 h to allow ILI formation, the cells were washed and re-incubated in fresh medium without lipoprotein but in the absence or presence of either THL or MmPPOX for an additional 24 h. The morphological appearance of the M. bovis BCG profiles was observed under the EM (Fig 5). In the absence of a lipase inhibitor, ILI +3 profiles were seldom encountered (Fig. 5A). In contrast, many ILI +3 profiles were encountered during a chase in presence of either THL (Fig. 5B) or MmPPOX (Fig. 5C). Scoring of the relative amount of each category of ILI profiles in each condition showed, as before [START_REF] Caire-Brandli | Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis[END_REF], that the percentage of ILI +3 profiles was reduced by 90% when cells were chased in medium without inhibitors (Fig. 5D). In contrast, when the chase medium contained either THL or MmPPOX approximately 80 to 90% of the ILI +3 profiles were retained (Fig. 5D). The fact that at 24 h the relative abundance of ILI +3 was 5 times higher in the presence of THL or MmPPOx (21% ± 0.6 and 23% ± 0.8, respectively) than in the absence of lipase inhibitors (3.6% ± 0.8) indicates that ILI consumption is indeed dependent on mycobacterial lipase activity. These results are in agreement with previous studies showing that hydrolysis of mycobacterial ILI is associated with the decrease of LB in the host [START_REF] Caire-Brandli | Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis[END_REF] and the presence of a (several) mycobacterial lipase(s) [START_REF] Dhouib | Watching intracellular lipolysis in mycobacteria using time lapse fluorescence microscopy[END_REF][START_REF] Low | Triacylglycerol utilization is required for regrowth of in vitro hypoxic nonreplicating Mycobacterium bovis bacillus Calmette-Guerin[END_REF], and more particularly LipY [START_REF] Kapoor | Human granuloma in vitro model, for TB dormancy and resuscitation[END_REF][START_REF] Deb | A novel lipase belonging to the hormone-sensitive lipase family induced under starvation to utilize stored triacylglycerol in Mycobacterium tuberculosis[END_REF][START_REF] Mishra | Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY[END_REF].

DISCUSSION

In previous work, VLDL was used as a source of lipids to generate FM [START_REF] Caire-Brandli | Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis[END_REF] since it is well known that such cells play a major role in the persistence and reactivation phases of TB within granulomas [START_REF] Peyron | Foamy macrophages from tuberculous patients' granulomas constitute a nutrient-rich reservoir for M. tuberculosis persistence[END_REF][START_REF] Hunter | Pathology of postprimary tuberculosis in humans and mice: contradiction of long-held beliefs[END_REF][START_REF] Kapoor | Human granuloma in vitro model, for TB dormancy and resuscitation[END_REF][START_REF] Russell | Foamy macrophages and the progression of the human tuberculosis granuloma[END_REF]. Quantitative analyses of detailed EM observations performed after exposure of M. avium-infected cells to VLDL had shown that macrophages became foamy and mycobacteria formed large ILI for which host TAG was essential. Lipid transfer occurred via mycobacterium-induced fusion between LB and phagosomes. This experimental tool clearly constitutes a well-defined cellular system in which to study changed metabolic states of intracellular mycobacteria that may relate to persistence and reactivation of TB. As a follow-up to this proof of concept, this FM model was used here to demonstrate the extensive accumulation of ILI inside M. bovis BCG and to investigate in detail the role of host lipases in LB formation, without which there can be no transfer of host TAG to mycobacterium-containing phagosomes, as well as the physiological role of the mycobacterial lipase, LipY, in the degradation of TAG inside mycobacterium-containing phagosomes and mycobacterial ILI. By a variety of approaches, we analyzed the contribution of macrophage and bacterial lipases in LB and ILI formation/degradation, and propose the following model depicted in Fig. 6.

After its internalization by receptor-mediated endocytosis, VLDL is transferred to lysosomes where host lysosomal lipases hydrolyze TAG into its derivatives and free fatty acid that serves to form LB. In a first set of experiments, we used two different families of lipase inhibitors, THL and MmPPOX, which, surprisingly, act by targeting different types of lipases within cells. On the one hand, by exposing infected cells to VLDL in the presence of THL, this lipase inhibitor shows preferences for host lipases, since TAG hydrolysis in lysosomes was blocked. Macrophages were unable to become foamy and, as a result, M. bovis BCG was unable to form ILI. On the other hand, exposure of cells to VLDL in presence of MmPPOX, blocked weakly the host lipases in the lysosomes and consequently did not profoundly affect the formation of LB, but mycobacteria still remained unable to form ILI. Our results suggest that the effect of this inhibitor is more specifically directed against mycobacterial lipases. The fact that these two families of inhibitors target lipases located at different sites could be directly related to their chemical structures and biophysical properties. In contrast to MmPPOX, THL is indeed a highly lipophilic molecule with physico-chemical properties similar to those of a diacylglycerol molecule which makes it soluble in VLDL. By using these two different families of lipase inhibitors, we can study either the host lipases or the mycobacterial lipases. We, therefore, provide strong evidence that ILI formation i) involves both host lysosomal and mycobacterial lipases, and ii) is strictly dependent on host LB in our experimental model.

In Dictyostelium discoideum, Mycobacterium marinum, was found to produce ILI without using TAG as the major carbon source [START_REF] Barisch | Lipid droplet dynamics at early stages of Mycobacterium marinum infection in Dictyostelium[END_REF][START_REF] Barisch | Mycobacterium marinum Degrades Both Triacylglycerols and Phospholipids from Its Dictyostelium Host to Synthesise Its Own Triacylglycerols and Generate Lipid Inclusions[END_REF]. Indeed, the use of a Dictyostelium knockout mutant for both Dgat enzymes (dgat1 and dgat2 genes), which is unable to produce LB, allowed the authors to demonstrate that a resulting excess of free fatty acids is predominantly incorporated into phospholipids triggering a massive ER-membrane proliferation. Fluorescent and EM approaches coupled with TLC lipid analyses suggested that M. marinum uses phospholipids to build-up ILI. This observation leads to a new hypothesis that intracellular mycobacteria have access to a wide range of host lipids, and one cannot exclude the possibility that bacteria in FM may have direct access to free fatty acids under conditions other than the experimental conditions of our FM model. Although this hypothesis has to be fully addressed experimentally in future studies, the current knowledge about ILI formation in mycobacteria within FM seems to predominantly rely on host-TAG degradation.

Among the large number of lipolytic enzymes encoded by the M. tuberculosis genome, LipY remains the only one carrying an N-terminal PE domain, which was subsequently found to be a signal of recognition by the ESX-5 secretion system [START_REF] Daleke | Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) protein domains target LipY lipases of pathogenic mycobacteria to the cell surface via the ESX-5 pathway[END_REF][START_REF] Daleke | General secretion signal for the mycobacterial type VII secretion pathway[END_REF].

Herein, we investigated whether the PE domain may directly participate in the enzyme's TAG activity. Our results are in agreement with a previous study demonstrating that LipY is the enzyme with the highest potential for hydrolyzing TAG stored inside M. tuberculosis [START_REF] Deb | A novel lipase belonging to the hormone-sensitive lipase family induced under starvation to utilize stored triacylglycerol in Mycobacterium tuberculosis[END_REF]. The authors extend our earlier report suggesting that the PE domain plays a role in modulating the catalytic activity of LipY [START_REF] Mishra | Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY[END_REF]. Comparison of the catalytic activities of rLipY and rLipY(ΔPE) clearly indicates that LipY(ΔPE) is more active than LipY, regardless of the substrate used. Similar results came from another group who used only one synthetic non physiological substrate [START_REF] Garrett | Modulation of the Activity of Mycobacterium tuberculosis LipY by Its PE Domain[END_REF]. Our data, therefore, add new insights into the functional role of the PE domain, which is shared by a large number of proteins in pathogenic mycobacteria. Interestingly, Mycobacterium marinum contains a protein homologous to LipY, termed LipYmar, in which the PE domain is substituted by a PPE domain [START_REF] Mishra | Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY[END_REF]. As for LipY, overexpression of LipYmar in M. smegmatis significantly reduced the TAG pool, and this was further pronounced when the PPE domain of LipYmar was removed, suggesting that PE and PPE domains can share a similar functional role. Therefore, given the analogy between LipY and LipYmar, it is conceivable to speculate that the PPE domain, like the PE domain, directly modulates the activity of LipYmar and that the lower catalytic activity of LipYmar may result from steric hindrance of the PPE domain, altering the recognition/accommodation of TAG within the lipid-binding site. Since, like M. tuberculosis or M. bovis BCG, M. marinum also produces ILI during infection (54) (and unpublished data), it is tempting to speculate that LipYmar plays a crucial role in ILI formation during M. marinum infection.

To further delineate the role of the PE domain in modulating the activity of LipY in an infectious context, experiments were carried out by extending our original experimental model of FM [START_REF] Caire-Brandli | Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis[END_REF]. BMDM were infected with M. bovis BCG strains overexpressing either LipY or LipY(ΔPE) and exposed to VLDL as a lipid source.

Quantitative analyses of EM observations allowed to perform a detailed investigation on the role of the mycobacterial lipase, LipY, in the degradation of host TAG transferred to the mycobacterium-containing phagosomes. Importantly, the difference in in vitro activity of LipY and LipY(ΔPE) could be reconciled in this model of infection where a significant reduction of the ILI +3 category was found with the BCG strain overexpressing LipY(ΔPE). This is consistent with the fact that, in contrast to LipY which is secreted, LipY(ΔPE) fails to be transported by ESX-5 and, as a consequence, its accumulation inside mycobacteria boosts ILI degradation [START_REF] Mishra | Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY[END_REF][START_REF] Abdallah | PPE and PE_PGRS proteins of Mycobacterium marinum are transported via the type VII secretion system ESX-5[END_REF].

In a previous study [START_REF] Caire-Brandli | Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis[END_REF], we had shown that removal of VLDL, induced a rapid decline of both LB and ILI. Here, we show that exposure of infected cells to lipase inhibitors, added during the chase period following an exposure to VLDL, strongly affects TAG hydrolysis within ILI, which remain abundant and large. These results provide evidence that cytosolic LipY is also involved in ILI consumption.

Our data also indicate that the dual localization of LipY impacts on its activity. Since the PE-containing domain is cleaved off by the ESX-5 secretion system [START_REF] Daleke | Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) protein domains target LipY lipases of pathogenic mycobacteria to the cell surface via the ESX-5 pathway[END_REF], our results suggest that the mycobacterial surface-anchored form of LipY is more active than the intracytosolic full-length protein. From these results, it can be inferred that LipY is more active against host lipids targeted to phagosomes via LB-phagosome fusion than towards TAG stored within ILI. Indeed, as illustrated in (Fig. 6), while a fraction of LipY is found in the cytosol, consistent with its role in the catabolism of intracellular TAG in ILI, a significant fraction of mature LipY lacking its PE domain is also localized at the outer surface of the mycobacterial cell envelope, where it catalyzes the breakdown of exogenously available TAG, such as those found within the LB of the FM. This scenario is consistent with the idea that M. tuberculosis depends on fatty acids as a preferred energy source during infection [START_REF] Munoz-Elias | Carbon metabolism of intracellular bacteria[END_REF] where LipY represents a critical enzyme for the utilization of host lipids. However, the ILI +3 profile in the mutant ∆lipY, clearly indicates the active participation of other mycobacterial lipases in this process, a finding consistent with the presence of a large array of lipolytic-encoding genes found in pathogenic mycobacteria [START_REF] Cole | Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence[END_REF]. Among these, Rv0183 has been shown to exhibit a preference for MAG or DAG and to be localized to the cell wall [START_REF] Dhouib | A monoacylglycerol lipase from Mycobacterium smegmatis Involved in bacterial cell interaction[END_REF][START_REF] Cotes | Characterization of an exported monoglyceride lipase from Mycobacterium tuberculosis possibly involved in the metabolism of host cell membrane lipids[END_REF], whereas Cfp21, a secreted cutinase-like enzyme also expresses TAG lipase activity [START_REF] Schué | Two cutinase-like proteins secreted by Mycobacterium tuberculosis show very different lipolytic activities reflecting their physiological function[END_REF][START_REF] West | Cutinase-like proteins of Mycobacterium tuberculosis: characterization of their variable enzymatic functions and active site identification[END_REF]. These two proteins may represent putative candidates which, in addition to LipY, may participate in the breakdown of host TAG transferred to phagosomes, and the release of free fatty acids which may be utilized by multiple TAG synthases [START_REF] Daniel | Induction of a novel class of diacylglycerol acyltransferases and triacylglycerol accumulation in Mycobacterium 39 tuberculosis as it goes into a dormancy-like state in culture[END_REF] to carry out the synthesis of TAG in the mycobacterial cytosol (Fig. 6). Utilization of these TAG in ILI is essential for the regrowth of mycobacteria during their exit from hypoxic non-replicating conditions [START_REF] Low | Triacylglycerol utilization is required for regrowth of in vitro hypoxic nonreplicating Mycobacterium bovis bacillus Calmette-Guerin[END_REF] and reactivation of latent infection to cause an active disease.

.

  DMEM and glutaraldehyde grade I (EM grade) were purchased from Sigma (St Louis, MO, USA), FBS was from Biowest (Nuaillé, France), PBS was from GIBCO (distributed by Invitrogen, Villebon sur Yvette, France), commercial Very Low Density Lipoprotein (VLDL) was from Calbiochem-Merck (Darmstadt, Germany), osmium tetroxide and Spurr resin were from Electron Microscopy Sciences (distributed by Euromedex, Mundolsheim, France). All bacterial culture media were purchased from Life technologies (USA). Bacterial strains and growth conditions. Escherichia coli DH10B cells (Invitrogen) used in cloning experiments were grown at 37°C in Luria Bertani (LB) broth (Invitrogen) or on LB agar plates. Culture media were supplemented with 200 µg/mL hygromycin B. The M. smegmatis mc 2 155 groEL1ΔC strain (30) used for expression experiments was routinely grown at 37°C with shaking (220 rpm) in Middlebrook 7H9 medium (Difco) supplemented with 0.05% Tween 80 (v/v), 0.2% glycerol (v/v), 0.5% bovine serum albumin (BSA) (w/v) and 0.2% glucose (w/v) or on Middlebrook 7H11 (Difco) agar plates.

Figure 1 :

 1 Figure 1: Addition of lipase inhibitors during exposure to VLDL affects host LB

Figure 2 :

 2 Figure 2: Purification and structural modelling of LipY and LipY(ΔPE). (A) Protein

Figure 3 :

 3 Figure 3: Distribution of ILI profiles in M. bovis BCG strains over-expressing

Figure 4 :

 4 Figure 4: LipY and additional mycobacterial lipases contribute to host TAG

Figure 5 :Figure 6 :

 56 Figure 5: Consumption of TAG within ILI correlates with the activity of cytosolic

Table 1 : 2 a

 12 Specific activities of rLipY and rLipY(ΔPE) on various lipid substrates a . All substrates were assayed at concentrations above their solubility limits. Experiments were performed at 37°C in 15 mL of 2.5 mM Tris-HCl buffer (pH 7.5) containing 300 mM NaCl and 1(a) or 3(b) mM NaTDC. * One unit corresponds to 1 µmole of fatty acid released per min. Values are means of 3 independent experiments ± SD.

Figure 1 952 953 Figure 6

 19536 Figure 1

ACKNOWLEDGMENTS

We are grateful to S. Rao (Novartis Institute for Tropical Diseases, Singapore) for the kind gift of the lipY deletion mutant. PS received financial support for his PhD fellowship from the Ministère Français de l'Enseignement Supérieur, de la Recherche et de l'Innovation. The authors wish to thank the support by the Fondation pour la Recherche Médicale (FRM) (DEQ20150331719) to LK and Campus France (Paris, France) for the PhD fellowship granted to SD. This work was supported by core grants from the Institut National de la Santé et de la Recherche Médicale (Inserm) and the Centre National de la Recherche Scientifique (CNRS), and by grant number ANR-09-MIEN-009-03 from the Agence Nationale de la Recherche (French National Research Agency) to LA., CdC., LK. and SC. The EM observations and analyses were performed by CdC and IP in the PiCSL EM core facility (Institut de Biologie du Développement, Aix-Marseille Université, Marseille), a member of the France-BioImaging French research infrastructure. This work has also benefited from the facilities and expertise of the Platform for Microscopy of IMM (Institut de Microbiologie de la Méditerranée). The authors wish to thank the members of these EM facilities for expert technical assistance, Jean Pierre Gorvel (Centre d'Immunologie de Marseille-Luminy, Aix-Marseille Université UM2, Inserm, U1104, CNRS UMR7280, Marseille, 13288, France) for continuous support and advice, Irène Caire-Brändli for expert technical assistance, she took part in the cellular microbiology experiments and EM observations and analyses under CdC's supervision. We would like to thank Pr Valéry Matarazzo and members from the INMED laboratory (INSERM-INMED UMR901, Aix-Marseille Université) for providing the femurs of 6-to 8-week-old C57BL/6 female mice. Finally, we kindly thank and Jean-François Cavalier (CNRS, UMR7255, Marseille, France) for providing the MmPPOX lipase inhibitor and for fruitful discussions.


Brice Le Borgne

Laurent Pichon
email: lpichon@univ-rennes1.fr

Anne Claire Salaun

Baptiste Le Bihan

Anne Jolivet- Gougeon

Sophie Martin

Regis Rogel

Olivier De Sagazan

Bacteria electrical detection using 3D silicon nanowires based resistor

Resistors based on silicon nanowires (SiNWs) are developed for the direct detection of Escherichia coli bacteria. The devices are manufactured using conventional silicon technology. The SiNWs are synthesized by the vapor liquid solid process (VLS) using gold as the catalyst. The electrodes of the device consist of highly in situ doped polycrystalline silicon. The results show that the corresponding resistances can be potentially used for the detection of bacteria by electrical measurements, using SiNWs as sensitive units. The bacteria are preferentially hooked in the SiNWs network, which causes a drastic reduction in the electrical resistance of the component. Such resistor manufactured in a simple and low-cost CMOS (Complementary Metal Oxide Semiconductor) technology acts as proof of concept. This device offers promising potentialities taking advantages of integration into CMOS

platform for alternative direct electrical detection of bacteria to monitor the contamination in environment for hygiene applications.

I. Introduction

One of the main challenges in terms of a public health is the rapid detection of bacteria in food, water and air, particularly because of the emergence of multi-resistant bacteria.

Although standard microbiological cell culture for identifying bacterial strains are well controlled, the detection process can often take several days. In addition, complex instrumentation [START_REF] Diggle | West Cooperation and conflict in quorum-sensing bacterial populations[END_REF] is required in most conventional methods, and cannot be used on site.

Classical methods for the detection of pathogens are polymerase chain reaction (PCR) [START_REF] Bej | Polymerase chain reaction-gene probe detection of microorganisms by using filter-concentrated samples[END_REF], colony culture and enumeration methods [START_REF] Leoni | Comparison of selective procedures for isolation and enumeration of Legionella species from hot water systems[END_REF], immunological methods [START_REF] Gu | Biofunctional magnetic nanoparticles for protein separation and pathogen detection[END_REF] and more recently detection of pathogens bacteria using mass spectrometry [START_REF] Shell | Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry as a reliable proteomic method for characterization of Escherichia coli and Salmonella isolates[END_REF]. The main drawbacks of these methods are significant analysis time, high cost equipment, and complexity of detection process.

The use of biosensors [START_REF] Deisingh | Biosensors for the detection of bacteria[END_REF] is an alternative method for detecting bacteria combining a biological recognition mechanism with a physical transduction technique. In this case transduction may be micromechanical, electrochemical, piezoelectric, thermometric, magnetic or optical. In particular, the electrochemical transduction methods such as amperometry [START_REF] Tang | A new amperometric method for rapid detection of Escherichia coli density using a self-assembled monolayerbased bienzyme biosensor[END_REF],

impedance spectroscopy [START_REF] Radke | A high density microelectrode array biosensor for detection of E. coli O157:H7[END_REF][START_REF] Radke | A microfabricated biosensor for detecting foodborne bioterrorism agents[END_REF][START_REF] Varshney | Interdigitated array microelectrode based impedance biosensor coupled with magnetic nanoparticle-antibody conjugates detection for Escherichia coli O157:H7 in food sample[END_REF], potentiometry [START_REF] Bergveld | Thirty years of ISFETOLOGY: What happened in the past 30 years and what may happen in the next 30 years[END_REF], are much faster and more sensitive than other techniques.

Bacteria detection systems are based on multidisciplinary fields requiring knowledge of physics, chemistry, biology, instrumentation, microelectronics and fabrication technology. In the objective of a lab on chip system for rapid detection of bacteria which may revolutionize health prevention, the challenge is the development of low cost and high sensitive new sensors that enable direct bacteria detection at low level. But none of the detection methods previously reported fulfils all these criteria. However, electrical detection of charged (bio)chemical species or polar molecules by measurement of the conductance change could be an alternative approach. Indeed, conductance variation, due to the charge effect facilitated by positioning species between two interconnected electrodes, preserves high sensitivity with less detection time [START_REF] Mahummad-Tahir | Alocilja A conductometric biosensor for biosecurity[END_REF]. In addition, based on this detection method, many groups have reported on field effect transistors (FET) used as biochemical sensors. In particular, chemical or biochemical (DNA hybridization) [START_REF] Cui | Nanowire nanosensors for highly sensitive and selective detection of biological and chemical species[END_REF][START_REF] Li | S Williams Sequence-Specific Label-Free DNA Sensors Based on Silicon Nanowires[END_REF], gas [START_REF] Chen | Nanowires based gas sensor[END_REF] or mechanical [START_REF] He | Giant piezoresistance effect in silicon nanowires[END_REF] sensors, have been demonstrated using silicon nanowires (SiNWs) based FETs. In this case nanowires act as sensing elements. However, few works report on SiNWs use as great promising method for label free, ultra-sensitive and real-time detection of bacteria [START_REF] Mishra | Ultrasensitive detection of bacterial toxin with silicon nanowire transistor Lab[END_REF][START_REF] Le Borgne | Silicon nanowires based resistors for bacteria detection[END_REF].

The recent progress in micro-and nano-technologies for fabrication of nano-objects compatible with silicon technology offer great opportunities for the development of innovative biosensors integrated on CMOS (Complementary Metal Oxide Semiconductor) platforms. These sensors could be interestingly directly coupled with any existing circuitry or signal conditioning systems to transmit their output data. Furthermore, due to their specific properties at nanoscale (high surface/volume ratio, carrier confinement), SiNWs are promising candidates as sensing elements for high sensitive bacteria detection at low level.

The main advantages of the use of such nano-objects are: the possibility of the mass production, the use of the volume of materials at low level, the micro-fluidic facilities for transport of reagents, the reduction of analysis time, and thus the reduced costs of fabrication and analysis.

However, the development of a small sized, reliable and versatile low-cost sensor based on nanowires has not been yet demonstrated. Indeed, many previous studies based on materials, structures and functionalization are reported into literature, but the proposed solutions do not fulfill the requirements for integration into a sensor. The major problem is the realization of such high-performance sensor with a low-cost technology, while the technological means used for the realization of nanowires require high cost and complex technologies based on ebeam lithography [START_REF] Li | S Williams Sequence-Specific Label-Free DNA Sensors Based on Silicon Nanowires[END_REF] or transfer techniques [START_REF] Lee | Kim Top-Down Fabrication of Fully CMOS-Compatible Silicon Nanowire Arrays and Their Integration into CMOS Inverters on Plastic[END_REF]. Nevertheless, alternative self-assembly methods [START_REF] Hochbaum | Controlled Growth of Si Nanowire Arrays for Device Integration[END_REF] are promising for synthesis of nanowires and do not require expensive lithographic tools.

Moreover, many chemical or biological silicon nanowires based sensors are issued from field effect transistor technology, but the manufacturing process includes many fabrication steps (at least four masks for lithography), with possible transfer techniques and sophisticated chemical steps for functionalization. On the other hand, the fabrication of sensors based on basic electronic devices (for instance resistors, capacitors), with direct integration of the nanowires as sensing elements, refers as simple and low-cost technology and constitutes a technological progress.

In this way, in this study the feasibility of SiNWs based resistor for bacteria detection with SiNWs used as sensing elements synthesized by vapour liquid solid (VLS) mechanism [START_REF] Hochbaum | Controlled Growth of Si Nanowire Arrays for Device Integration[END_REF] (self-assembly method) is investigated. Such device acts as proof of concept and fulfills the following requirements: i) low cost technology, ii) high sensitivity, iii) real-time analysis, iv) electrical detection of bacteria, and v) portability and on site measurements.

II. Material and method

Devices

The resistors used for bacteria detection applications (fig. 1) are fabricated in a classical silicon technology following a two masks process. Comb shape inter-digitated electrodes of the devices are made of a highly phosphorous in-situ doped amorphous silicon layer deposited by Low Pressure Chemical Vapour Deposition (LPCVD) technique at 550°C, and solid phase crystallized by thermal annealing at 600°C under vacuum to get a highly in-situ doped polycrystalline silicon (polySi). Silicon nanowires (sensitive units) are locally grown on the teeth of the polySi inter-digitated electrodes by the well-known VLS mechanism at 460°C using gold as catalyst. In this way no patterning technique was used for elaboration of nanowires. The diameter and length of the SiNWs are 100nm and more than 20µm respectively. Nanowires bridge between two adjacent teeth and thus ensure electrical connection between the two highly doped polySi electrodes (fig. 1. (b)). The potential use as high sensitive gas (ammonia) sensors of such SiNWs based resistors was previously reported [START_REF] Demami | Silicon nanowires based resistors as gas sensors[END_REF]. Escherichia coli ATCC 35218 bacteria deposition was carried out from serial dilutions in sterile distilled water of an overnight bacterial culture in trypticase soy broth (BBL Trypticase Soy Broth, Becton Dickinson®) incubated at 37°C. This culture was adjusted to 8×10 8 CFU/mL by measurement of OD with spectrophotometer at 600 nm (OD 600 = 0.6). Then, a serie of dilutions of bacteria in sterile distilled water was performed from 8×10 8 to 8×10 3 CFU/mL. Bacterial counts were verified by plating each dilution (100µL) on trypticase agar plates (Oxoid®), and enumerating the colonies after 24h of incubation at 37°C. Escherichia coli K.12 bacteria deposition was carried out from serial dilutions in sterile distilled water of an overnight bacterial culture in LB broth Lennox (Difco) incubated at 37°C. This culture was adjusted to 6×10 8 CFU/mL by measurement of OD with spectrophotometer at 600 nm, OD = 0,1. Then, a serie of dilutions of bacteria was performed from 6×10 8 to 6×10 4 CFU/mL. After bacteria solution deposition (30µL), devices were dried at room temperature and tested 24h later to avoid any moisture effect on the electrical behaviour of the resistor. Note that, considering the size of the device, a 2µL droplet could be used to limit the waste of material and to drastically decrease the drying time. It would consequently significantly reduce the response time of the sensor from 24h to less than one hour. In addition, some devices submitted to a mixture of sterile distilled water and culture solution without any bacteria were used as references.

Bacteria detection was checked by measuring static I-V curves of the SiNWs based resistors using an Agilent B1500A semiconductor parameter analyser. Measurements were performed at room temperature on devices based on n = 20, 40, and 100 teeth of the comb shape electrode, with each tooth spaced from W = 5µm.

III. Results and discussion

Because of the SiNWs VLS process growth, for a fixed value of W, the number of interconnected nanowires (conducting paths) between two adjacent N-type polySi teeth can change. In addition, the sizes of the nanowires are distributed over an average value, which means that the corresponding elementary resistance value can fluctuate. Thus, it might play a role in the electrical resistance measurements between two adjacent N + polySi electrodes.

However, in order to simplify and because of the high number of the nanowires we assume that electrical and geometrical characteristics are identical for all interconnected nanowires. In this case, these elementary resistances, R 0, are assumed to be identical. Thus the equivalent electrical circuit of the whole resistor represented in the figure 2 results to 2n elementary resistances connected in parallel. Therefore, the total resistance of the resistor can be expressed as the following formula:

n R I V R 2 0 ≈ = (1)
In these conditions, macroscopic theoretical model for calculation of R 0 has been previously reported by our group to estimate the average electrical resistivity of a single undoped SiNW (10 6 -10 7 Ω cm) [START_REF] Pichon | Electrical properties of phosphorus in-situ doped Aucatalyst vapor liquid solid silicon nanowires[END_REF]. In this model R 0 results from interconnections of nanowires and is related to the average electrical resistivity of the nanowire, ρ NW , following:

K R NW × = ρ 0 (2)
with K the geometrical factor depending of the total conducting paths. The high value reported of the average electrical resistivity of a single nanowire for undoped SiNWs is related to carriers trapping effect at defects located at interconnections between nanowires and induced by gold impurities at the surface of the nanowires. (for an average value R 0 ≈1.9×10 9 Ω), and validate the equivalent electrical model given by [START_REF] Diggle | West Cooperation and conflict in quorum-sensing bacterial populations[END_REF].

Interaction of bacteria with the surface of materials is mainly controlled by the charge effects and hydrophobic properties between bacteria and materials [START_REF] Neu | Bacterial polymers: physicochemical aspects of their interactions at interfaces[END_REF]. In particular, the adhesion can occur when the van der Waals forces of attraction between the membrane of the bacteria and the surface of the material outweigh the electrostatic repulsion forces [START_REF] Marshall | Adsorption and adhesion process in microbial growth at interfaces[END_REF][START_REF] Rijnaarts | Reversibility and mechanism of bacterial adhesion[END_REF]. After bacteria immobilization on the device, bacteria interaction with SiNWs is firstly analysed by SEM observations (fig. 4). The bacteria optical count has not been performed in this study. Interestingly, in our case, pictures clearly show that bacteria are mainly attached into the silicon nanowires mesh rather than in other parts of the device. This observation shows the interest to use a 3D SiNWs tangled mesh to promote bacteria immobilization and thus detection. Functionalization of active parts for detection, usually based on sophisticated chemical steps, is a common method to allow immobilization and identification of chemical of biochemical species [START_REF] Lu | Yew Bacteria detection utilizing electrical conductivity[END_REF]. However, in our case families of bacteria cannot be identified.

Voltage (V) (a) Escherichia coli K 12 bacteria -6 -4 -2 0 2 4 6 -1,0x10 -6 -8,0x10 -7 -6,0x10 -7 -4,0x10 -7 -2,0x10 -7 0,0 2,0x10 -7 4,0x10 -7 6,0x10 -7 8,0x10 -7 1,0x10 -6 6
Voltage (V)

. 10 8 CFU/ml 6. 10 7 CFU/ml 6. 10 5 CFU/ml 6. 10 4 CFU/ml No bacteria Current (A) measurements performed on SiNWs based resistors. As shown in the figure 5, for the two types of bacteria I-V curves exhibit a drastic increase of the current corresponding to a lowering of the R value of the resistor due to the presence of bacteria. This phenomenon is probably due to the negative charge of their peptidoglycan (PG) based membrane [START_REF] Lu | Yew Bacteria detection utilizing electrical conductivity[END_REF][START_REF] Gottenbos | Antimicrobial effects of positively charged surfaces on adhering Gram-positive and Gramnegative bacteria[END_REF].

Voltage (V) (f)
Indeed, two main types of bacteria are listed related to their membrane: Gram positive (+) and

Gram negative (-) bacteria. For the Gram-positive bacteria, membrane contains a thick (20-50 nm) PG layer attached to teichoic acids [START_REF] Scott | Surface proteins of gram-positive bacteria and how they get there[END_REF]. In the case of Gram-negative bacteria as E.

Coli, the membrane is made of a thin PG layer covered by an extern membrane containing lipopolysaccharides components, responsible of a higher negative charge of the bacteria [START_REF] Roberts | The biochemistry and genetics of capsular polysaccharide production in bacteria[END_REF].

To explain the decrease of the global electrical resistance due to the capture of bacteria three effects can be invoked. One first explanation could be a transfer of charges (electrons) of the bacteria into the nanowires which fill the traps previously mentioned, and thus could and contribute to the decrease of the electrical resistivity of the nanowires [START_REF] Pichon | Electrical properties of phosphorus in-situ doped Aucatalyst vapor liquid solid silicon nanowires[END_REF]. Another possible reason is that the electrically charged bacteria may behave as biological gate resulting as wellknown field effect [START_REF] Cui | Nanowire nanosensors for highly sensitive and selective detection of biological and chemical species[END_REF][START_REF] Li | S Williams Sequence-Specific Label-Free DNA Sensors Based on Silicon Nanowires[END_REF][START_REF] Chen | Nanowires based gas sensor[END_REF][START_REF] Mishra | Ultrasensitive detection of bacterial toxin with silicon nanowire transistor Lab[END_REF] on the SiNWs. At last, other explanation is that bacteria may act as additional conducting paths randomly distributed between teeth thanks to hooking effect of bacteria into the SiNWs network. This phenomenon would contribute to the variations of R 0 values, ∆R, strongly dependent on the number of bacteria hanged between two adjacent teeth (see fig. 6). Based on these three considerations, further investigations for a well understanding of the carrier transport in these devices in relation with bacteria and nanowires interactions must be conducted for consideration in the model given by (2). Escherichia coli K.12. Grey area: detection range value

Because the performances of a bacteria sensor are strongly related to its abilities for quantitative recognition of target analytes, further studies in function of the size, the quantity, and the type of targeted bacteria have to be carried out for applications of our SiNWs based resistors as selective bacteria sensors. Nevertheless, sensing properties of our SiNWs based resistors have s low detection limit, working range and time analysis to many others bacteria detection techniques (Enzyme Linked ImmunoSorbent Assay-ELISA, PCR, QCM-Quartz Crystal Microbalance immunosensor, impedimetric, amperometric…) [START_REF] Lazcka | Pathogen detection : A perspective of traditional methods and biosensors[END_REF][START_REF] Ahmed | Biosensors for whole-cell bacteria detection[END_REF].

IV. Conclusion

In this work, investigations on a SiNWs based resistor were carried out to study the potential development of a sensitive real-time detection bacteria sensor, fabricated by a simple low-cost fabrication and compatible CMOS silicon technology, using VLS SiNWs as sensitive elements. Such a technology takes advantages of both benefits at nanoscale and microtechnology for miniaturization and integration on microsystems, and of the use of SiNWs network to favour bacteria hanging to increase the sensitivity for detection of bacteria.

The detection is based on the electrical measurement. The resistor showed its ability for bacteria detection (Escherichia coli ATCC 35218 and K12) at concentration as low as 6x10 3 CFU/mL. Since the change into electrical resistances values of the device were not correlated with bacteria concentrations, it could be used only for yes/no diagnosis. Further studies are needed to use the SiNWs based resistor as quantitative detection system and at lower detection limit. However, because such device is easy to use, reagent-less, and portable, it has the potential to be used as further detection tool for biosafety.

Figure 1 :

 1 Figure 1: VLS SiNWs based resistor: schematic view (a). SEM top view observation (b).

W

Figure 2 :

 2 Figure 2: Equivalent circuit diagram of SiNWs based resistors

9 ΩFigure 3 :

 93 Figure 3: I-V characteristics of the SiNWs based resistors made of interdigitated comb

Figure 4 :

 4 Figure 4: SEM pictures of bacteria (dark areas) hanged into SiNWs network of the resistor

Figure 5 :

 5 Figure 5: I-V curves of the SiNWs based resistors with n = 20, 40, 100 teeth and 5 µm

Figure 6 :Figure 7 :

 67 Figure 6: Illustration of the electrical resistance change with bacteria interaction