Gallois Nicolas

Alpha-Bazin Béatrice

Ortet Philippe

Barakat Mohamed

Piette Laurie

Long Justine

Berthomieu Catherine

Armengaud Jean
email: jean.armengaud@cea.fr

# Chapon

Proteogenomic insights into uranium tolerance of a Chernobyl's Microbacterium bacterial isolate

Keywords: Radionuclides, toxicity, proteogenomics, bioinformatics, toxicoproteomics, Microbacterium

Microbacterium oleivorans A9 is a uranium-tolerant actinobacteria isolated from the trench T22 located near the Chernobyl nuclear power plant. This site is contaminated with different radionuclides including uranium. To observe the molecular changes at the proteome level occurring in this strain upon uranyl exposure and understand molecular mechanisms explaining its uranium tolerance, we established its draft genome and used this raw information to perform an in-depth proteogenomics study. High-throughput proteomics were performed on cells exposed or not to 10 µM uranyl nitrate sampled at three previously identified phases of uranyl tolerance. We experimentally detected and annotated 1,532 proteins and highlighted a total of 591 proteins for which abundances were significantly differing between conditions. Notably, proteins involved in phosphate and iron metabolisms show high dynamics. A large ratio of proteins more abundant upon uranyl stress, are distant from functionally-annotated known proteins, highlighting the lack of fundamental knowledge regarding numerous key molecular players from soil bacteria.

Biological significance

Microbacterium oleivorans A9 is an interesting environmental model to understand biological processes engaged in tolerance to radionuclides. Using an innovative proteogenomics approach, we explored its molecular mechanisms involved in uranium tolerance. We sequenced its genome, interpreted high-throughput proteomic data against a six-reading frame ORF database deduced from the draft genome, annotated the identified proteins and compared protein abundances from cells exposed or not to uranyl stress after a cascade search. These data show that a complex cellular response to uranium occurs in Microbacterium oleivorans A9, where one third of the experimental proteome is modified. In particular, the uranyl stress perturbed the phosphate and iron metabolic pathways. Furthermore, several transporters have been identified to be specifically associated to

Microbacterium oleivorans A9 chromosome

Contig7_31611_-3 Contig7_33510_-3 Contig7_34548_-3 Contig7_35796_-3 Contig7_36731_-2 Contig7_38967_-3 Contig7_32419_-1

uranyl stress, paving the way to the development of biotechnological tools for uranium decontamination.

Introduction

Radionuclides (RNs) are naturally present in the environment but are redistributed by anthropogenic activities e.g. chemical nitrogen fertilizer use, weapons manufacturing, nuclear research and nuclear fuel production. Several major nuclear accidents such as Chernobyl and Fukushima led to the dispersion of several RNs through the atmosphere in microparticles [START_REF] Abe | Detection of uranium and chemical state analysis of individual radioactive microparticles emitted from the Fukushima nuclear accident using multiple synchrotron radiation X-ray analyses[END_REF] and in the soil [START_REF] Bugai | Characterization of subsurface geometry and radioactivity distribution in the trench containing Chernobyl clean-up wastes[END_REF]. In Chernobyl, radionuclides-contaminated wastes were buried on site in about 800 trenches. Besides 137 Cs and 90 Sr, other RNs are present in the Chernobyl trench T22, including 60 Co, 154 Eu, 238, 239, 240 Pu, 241 Am and 235, 238 U.

Uranium (U) belongs to the actinide series. It exhibits both radiological and chemical toxicities, and a long half-life, resulting in environmental and human health major concern [START_REF] Cothern | Health effects guidance for uranium in drinking water[END_REF]. As a hard Lewis acid and according to the HSAB theory which qualitatively explains the stability of metal complexes [START_REF] Pearson | Hard and Soft Acids and Bases[END_REF],

uranyl tends to interact with hard Lewis base such as phosphate, sulfate, carbonate or amine by ionic interactions. It can displace other hard Lewis acids like Ca 2+ and Fe 3+ in highly oxygenated site.

Moreover, different studies have confirmed the affinity between uranium and phosphate or carbonate [START_REF] Reeder | Coprecipitation of uranium(VI) with calcite: XAFS, micro-XAS, and luminescence characterization[END_REF][START_REF] Renninger | Uranyl precipitation by Pseudomonas aeruginosa via controlled polyphosphate metabolism[END_REF][START_REF] Yong | Role of citrate as a complexing ligand which permits enzymically-mediated uranyl ion bioaccumulation[END_REF][START_REF] Merroun | Bacterial interactions with uranium: an environmental perspective[END_REF][START_REF] Nakajima | Studies on the Accumulation of Heavy-Metal Elements in Biological-Systems .5. Ion Effects on the Uptake of Uranium by Chlorella-Regularis[END_REF], or the replacement of iron by uranyl [START_REF] Bouby | Complexation of uranium VI with the siderophore pyoverdine[END_REF].

Bacteria can interact directly or indirectly with RNs and change their speciation, playing a major role in their mobility and transfer in the environment. The use of both molecular-and culture-based approaches to study bacterial diversity in the trench T22 demonstrated that RNs-contaminated soils host an unexpected diversity of bacteria [START_REF] Chapon | Microbial diversity in contaminated soils along the T22 trench of the Chernobyl experimental platform[END_REF].

During their sampling campaign at Chernobyl, Chapon and colleagues have constructed a large collection of cultured bacteria from the trench T22 soils offering the opportunity to study the RN tolerance of these bacteria [START_REF] Chapon | Microbial diversity in contaminated soils along the T22 trench of the Chernobyl experimental platform[END_REF]. The strain Microbacterium oleivorans A9 (referred as Microbacterium sp. A9_sp3_-1_2 in [START_REF] Theodorakopoulos | Use of combined microscopic and spectroscopic techniques to reveal 20 interactions between uranium and Microbacterium sp. A9, a strain isolated from the Chernobyl exclusion zone[END_REF]) is one of the most uranium-tolerant isolate of this collection. In a previous paper, the interactions between uranium and this bacterium have been investigated and specific exposure conditions in which the bacteria were kept alive while being exposed to soluble forms of uranium have been set-up. With this tightly controlled exposure conditions, it has been shown that Microbacterium oleivorans A9 exhibit three sequential mechanisms involved in uranium detoxification: a rapid metal removal within the first 30 minutes, then an active U(VI) release in the exposure medium accompanied by a phosphate efflux and a final biomineralization step of uranium in autunite-like mineral phases [START_REF] Theodorakopoulos | Use of combined microscopic and spectroscopic techniques to reveal 20 interactions between uranium and Microbacterium sp. A9, a strain isolated from the Chernobyl exclusion zone[END_REF]. For this reason, it constitutes a relevant model to study the mechanisms involved in uranium tolerance and in uranium-bacteria interactions. In bacteria, survival in metal-contaminated environment is mainly achieved through active efflux pumps [START_REF] Nies | Microbial heavy-metal resistance[END_REF]. If such systems are well-known for a wide variety of non-essential metals, involvement of an efflux system in detoxification of uranium has not yet been described, although up-regulation of genes encoding metal efflux pumps has already been reported for Desulfotomaculum reducens exposed to U(VI) in anaerobic conditions [START_REF] Junier | The Response of Desulfotomaculum reducens MI-1 to U(VI) Exposure: A Transcriptomic Study[END_REF].

The cellular response of Escherichia coli, Acidithiobacillus ferrooxidans and two Anabaena strains upon uranium exposure has already been studied with 2D-PAGE based proteomic methodology [START_REF] Khemiri | Escherichia coli response to uranyl exposure at low pH and associated protein regulations[END_REF][START_REF] Dekker | Comparative proteomics of Acidithiobacillus ferrooxidans grown in the presence and absence of uranium[END_REF][START_REF] Panda | Proteomic analysis reveals contrasting stress response to uranium in two nitrogen-fixing Anabaena strains, differentially tolerant to uranium[END_REF]. These studies highlighted an impact of uranium on the bacterial proteome, but the low throughput of this approach allowed identifying only 11, 18, 45 and 27 proteins respectively. These may represent only a partial view of the proteins modulated upon uranium stress. Two studies using high-throughput tandem mass spectrometry have deepened the proteome changes occurring in two other bacteria, Geobacter sulfurreducens and Caulobacter crescentus [START_REF] Orellana | Proteome of Geobacter sulfurreducens in the presence of U(VI)[END_REF][START_REF] Yung | Shotgun proteomic analysis unveils survival and detoxification strategies by Caulobacter crescentus during exposure to uranium, chromium, and cadmium[END_REF]. These papers show that resistance to uranium is a complex cellular response with induction of multiple stress response systems. These four studies, based on gram-negative organisms, have used different uranium concentrations (from 50 to 500 µM) as well as different exposure conditions making any comparison difficult.

Proteogenomics, the alliance of genomics and proteomics, has proved its efficiency in supporting the existence of genes encoding hypothetical proteins or with unknown function, and even unannotated in the genome for several organisms [START_REF] Armengaud | Next-generation proteomics faces new challenges in environmental biotechnology[END_REF]. This methodology is even more appropriate when a quick focus on mechanisms for which the main players are unknown is required [START_REF] De Groot | Alliance of proteomics and genomics to unravel the specificities of Sahara bacterium Deinococcus deserti[END_REF][START_REF] Rubiano-Labrador | Proteogenomic insights into salt tolerance by a halotolerant alpha-proteobacterium isolated from an Andean saline spring[END_REF][START_REF] Zhu | Tissue-specific proteogenomic analysis of Plutella xylostella larval midgut using a multialgorithm pipeline[END_REF]. Because, proteogenomics is based on the assignment of MS/MS spectra with a six-frame translation of the genome, time-consuming, resource-dependent and hard-working genome sequence completion steps are bypassed. For example, the annotation of Deinococcus deserti by means of proteogenomics has shown its added value to correct annotation errors in other Deinococcus sp. genomes [START_REF] De Groot | Alliance of proteomics and genomics to unravel the specificities of Sahara bacterium Deinococcus deserti[END_REF][START_REF] Baudet | Proteomics-based Refinement of Deinococcus deserti Genome Annotation Reveals an Unwonted Use of Non-canonical Translation Initiation Codons[END_REF].

The exploration of the halotolerance molecular mechanisms of Tistlia consotensis by proteogenomics based on a draft genome sequence straightforwardly highlighted major changes in response to hypoosmotic and hyper-osmotic conditions [START_REF] Rubiano-Labrador | Proteogenomic insights into salt tolerance by a halotolerant alpha-proteobacterium isolated from an Andean saline spring[END_REF][START_REF] Rubiano-Labrador | Salt Stress Induced Changes in the Exoproteome of the Halotolerant Bacterium Tistlia consotensis Deciphered by Proteogenomics[END_REF].

Here, the molecular changes occurring in Microbacterium oleivorans A9 upon uranium exposure were deciphered by means of a proteogenomics approach consisting in draft genome sequencing and high-throughput proteome coverage by next-generation proteomics. Proteomic changes of cells exposed to 10 µM uranyl for 30 min, 4 h and 24 h were investigated. This experimental strategy allowed identification without a priori of soluble and membrane proteins with significantly differing abundances compared to controls, which could be players of the defense mechanism of Microbacterium oleivorans A9 against uranium. Furthermore, these genome and proteome datasets represent invaluable resources to gain insights into the physiology and role of Microbacterium in soils.

Experimental procedures

Uranium exposure

The experimental procedure used to expose the bacteria to uranium is described in [START_REF] Theodorakopoulos | Use of combined microscopic and spectroscopic techniques to reveal 20 interactions between uranium and Microbacterium sp. A9, a strain isolated from the Chernobyl exclusion zone[END_REF].

Microbacterium oleivorans A9 strain was routinely cultivated in 0.1 X Tryptic Soy Broth (TSB, Difco Laboratories) at 30°C with shaking. Cells at exponential growth phase were harvested by centrifugation for 10 min at 5000 g. From this stage on, samples were maintained at 25°C throughout the experiment. The resulting cell pellets were washed twice in 0.1 M NaCl pH 5.0 and were resuspended at about 6 x 10 9 cells mL -1 in 0.1 M NaCl pH 5.0 with 0 (control) or 10 µM U(VI). U(VI) was added as uranyl nitrate UO 2 (NO 3 ) 2 .6H 2 O (Sigma-Aldrich) from a 7.51 mM stock solution in 16 mM HNO 3 . No precipitate was observed along the exposure conditions. The nitrate concentration was adjusted to 0.416 mM by adding NaNO 3 when needed. Bacteria exposed to U(VI) and controls were incubated at 25°C with shaking. Four independent biological replicates were made for each condition and for each time point. Fractions of 1 ml of cell suspension were taken after 0.5, 4, and 24 h of uranium exposure. Samples were centrifuged for 5 min at 8000 g. The resulting supernatants were removed and the pellets were conserved at -80 °C until proteomic analysis.

Genome of Microbacterium oleivorans A9

Genomic DNA was sequenced on a HiSeq 2000 sequencing platform (Illumina) by the GenoScreen Company and genome de novo assembling was performed on the reads using ABYSS [START_REF] Simpson | ABySS: a parallel assembler for short read sequence data[END_REF]. The Whole Genome Shotgun project data have been deposited at DDBJ/EMBL/GenBank under the accession number MTIO00000000. For each contig, the ORF translated sequences were extracted from stop codon to stop codon in each of the six possible reading frames. The ORF-derived polypeptide list was restricted to those exhibiting a length of at least 50 amino acids. Proteomic data and proteogenomic procedures were then used to discriminate between false, correct but undetected, and correct and detected protein sequences as described earlier [START_REF] De Groot | Alliance of proteomics and genomics to unravel the specificities of Sahara bacterium Deinococcus deserti[END_REF][START_REF] Armengaud | Microbial proteogenomics, gaining ground with the avalanche of genome sequences[END_REF]. The ORF sequences for which at least one spectrum has been assigned were kept to create a second database. This step allowed us to identify more proteins and to improve qualitatively and quantitatively the MS/MS spectra attribution. The protein sequences were given a cluster of orthologous groups (COG) assignment based on the reference database [START_REF] Tatusov | The COG database: a tool for genome-scale analysis of protein functions and evolution[END_REF]. Furthermore, enzymes annotation using PRIAM profiles [START_REF] Claudel-Renard | Enzyme-specific profiles for genome annotation: PRIAM[END_REF] associated to KEGG database [START_REF] Kanehisa | KEGG as a reference resource for gene and protein annotation[END_REF] allowed the identification of metabolic pathways. An in-house batch program was developed to emphasize metabolic pathways highlighted by proteomic analysis.

Proteome sample preparation and trypsin in-gel proteolysis

Cell pellets (between 2.0 and 4.6 mg, 3.1 mg average, wet weight) were dissolved in a given volume (100 μL for 1.7 mg of bacterial pellet) of 1X NuPAGE TM LDS Sample Buffer (Invitrogen) supplemented with 2% β-mercaptoethanol. Samples were sonicated for 5 min in a transonic 780H sonicator and subjected to a 5 min incubation at 99°C prior to SDS-PAGE. Samples were subjected to a 3 min short migration in denaturing conditions as previously described [START_REF] Hartmann | Taking the shortcut for highthroughput shotgun proteomic analysis of bacteria[END_REF]. For this, samples were loaded onto a under vacuum as before. The gel bands were then processed for in-gel proteolysis with trypsin in presence of 0.01% ProteaseMax (Promega) as previously described [START_REF] Clair | Expanding the known repertoire of virulence factors produced by Bacillus cereus through early secretome profiling in three redox conditions[END_REF].

NanoLC-MS/MS analysis

NanoLC-MS/MS experiments for the 24 resulting peptide mixtures were performed using a Q-Exactive HF mass spectrometer (ThermoFisher) coupled to an UltiMate 3000 LC system (Dionex-LC Packings) in similar conditions as those previously described [START_REF] Klein | RNA-binding proteins are a major target of silica nanoparticles in cell extracts[END_REF]. Peptide mixtures (10 μl) were loaded and desalted on-line on a reverse phase precolumn (Acclaim PepMap 100 C18, 5 μm bead size, 100 Å pore size, 5 mm × 300 μm id) from LC Packings. Peptides were then resolved onto a reverse phase Acclaim PepMap 100 C18 column (3 µm, 100 Å, 500 mm x 75 µm id) at a flow rate of 0.3 μl/min with a 180 min linear gradient of CH 3 CN/0.1% formic acid and injected into the Q-Exactive HF mass spectrometer. The Q-Exactive HF instrument was operated according to a Top20 datadependent acquisition method as previously described [START_REF] Klein | RNA-binding proteins are a major target of silica nanoparticles in cell extracts[END_REF]. Briefly, a scan cycle was initiated with a full scan of peptide ions in the ultra-high-field Orbitrap analyzer, followed by selection of a single precursor and its dissociation in high energy collisional mode, and MS/MS scans on the 20 most abundant precursor ions. Full scan mass spectra were acquired with an Automatic Gain Control Target set at 3x10 6 ions and a resolution of 60,000 from m/z 350 to 1,800. MS/MS scan was initiated at a resolution of 15,000 when the ACG target reached 1x10 5 ions with a threshold intensity of 83,000 and potential charge states of 2+ and 3+. Precursor ions were selected with a dynamic exclusion of 10 sec for increasing the reliability of spectral count measurements.

Interpretation of mass spectrometry data

The recorded MS/MS spectra for the 24 samples were merged after being searched against the home-made ORF database. First, peak lists were generated with the MASCOT DAEMON software (version 2.3.2) from Matrix Science using the extract_msn.exe data import filter from the Xcalibur FT package (version 2.0.7) proposed by ThermoFisher. Data import filter options were set as previously described [START_REF] Christie-Oleza | Proteomic insights into the lifestyle of an environmentally relevant marine bacterium[END_REF] at 400 (minimum mass), 5000 (maximum mass), 0 (grouping tolerance), 0 (intermediate scans), and 1000 (threshold). MS/MS spectra were searched with MASCOT against the ORF database with the following parameters: tryptic peptides with a maximum of 2 miss cleavages during proteolytic digestion, a mass tolerance of 5 ppm on the parent ion and 0.02 Da on the MS/MS, fixed modification for carbamidomethylated Cys (+57.0215) and variable modification for oxidized Met (+15.9949). All peptide matches with a peptide score above its query threshold set at p ≤ 0.05 with the ORF database and rank 1 were parsed using the IRMa 1.31.1c software [START_REF] Dupierris | A toolbox for validation of mass spectrometry peptides identification and generation of database: IRMa[END_REF]. MS/MS spectra assigned to several loci were systematically removed. The average error for the determination of peptide mass is 0.59 ppm and the mean MASCOT score is 50.7. A protein was considered validated when at least two different peptides were detected. False-positive identification of proteins was estimated using a reverse decoy database as below 0.1% with these parameters.

The mass spectrometry proteomics data have been deposited in a public repository at the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [START_REF] Cote | The PRoteomics IDEntification (PRIDE) Converter 2 framework: an improved suite of tools to facilitate data submission to the PRIDE database and the ProteomeXchange consortium[END_REF] with the dataset identifier PXD005794 and project DOI 10.6019/PXD005794.

The number of MS/MS spectra per protein (spectral counts) was determined for the four replicates of each of the three time points for both conditions. The protein abundances were compared for each time point between the uranyl exposure and the control conditions. For each of these comparisons, the list of non-redundant proteins detected among the six corresponding datasets was for each protein was calculated using the formula: (spectral counts / theoretical molecular weight) x 1000, see [START_REF] Christie-Oleza | Proteomic insights into the lifestyle of an environmentally relevant marine bacterium[END_REF].

Results and discussion

Microbacterium oleivorans A9 genome annotation

The main characteristics of Microbacterium oleivorans A9 genome is described in [START_REF] Ortet | Draft genome sequence of Microbacterium oleivorans strain A9, a bacterium isolated from Chernobyl radionuclide-contaminated soil[END_REF]. Briefly, the genome size is around 2.95 Mbp. The G+C content of the genome is 68.33%. COG assignment for the resulting polypeptide dataset shows that two classes are predominant: proteins involved in metabolism of amino acids (74 proteins) on the first hand, and those involved in metabolism of carbohydrates (66 proteins) on the other. A search against the BacMet database [START_REF] Pal | BacMet: antibacterial biocide and metal resistance genes database[END_REF], which comprises antibacterial biocides and metal resistance genes, highlights the presence of 104 unique metal resistance genes such as actP gene which encodes a copper-transporting P-type ATPase, arsB gene which encodes an arsenic pump membrane protein or yieF gene which encodes a chromate reductase. We found a 16S rRNA gene copy in the genome, exhibiting 99% sequence identity with Microbacterium oleivorans 16S rRNA gene. Moreover, the assembling results are strikingly similar to those obtained for Microbacterium oleivorans strain RIT293 (RefSeq NZ_JFYO00000000.1) [START_REF] Gan | Whole-genome sequences of 13 endophytic bacteria isolated from shrub willow (salix) grown in geneva, new york[END_REF] with a median total length of 2.9 Mbp, a G+C content of 69% and a total of 2,732 protein-coding genes. This genome version comprises 11 contigs with a N50 parameter at 467,109 bp.

Shotgun proteogenomics of Microbacterium oleivorans A9

Proteogenomics quick panorama

Figure 1 shows the strategy applied here for quickly deciphering the proteome dynamics from resting cells exposed or not to 10 µM uranium. A total of S1). Because of the unusual large size of the six reading frame ORF database(30,853 putative polypeptide sequences instead of 2813 annotated coding DNA sequences), a second round of search was initiated after reducing the database to the proteins detected in the first round as proposed previously for improving the sensitivity of proteogenomics studies [START_REF] Jagtap | A two-step database search method improves sensitivity in peptide sequence matches for metaproteomics and proteogenomics studies[END_REF]. The second search led to the assignation of 747,621 peptide-to-spectrum matches, certifying the detection of 17,027 unique peptide sequences and 1,532 non-redundant proteins identified with at least two different peptides (Supplementary data Table S2). This two-round search allows increasing the number of proteins validated with at least two different peptides (1532 instead of 1504) and enhances the number of assigned MS/MS spectra (747 621 instead of 746 092). This dataset compares favorably with previous studies of bacteria exposed to uranium, as a dataset of less than 950 proteins were reported from Caulobacter crescentus [START_REF] Yung | Shotgun proteomic analysis unveils survival and detoxification strategies by Caulobacter crescentus during exposure to uranium, chromium, and cadmium[END_REF] and 1,363 proteins were listed from Geobacter sulfurreducens [START_REF] Orellana | Proteome of Geobacter sulfurreducens in the presence of U(VI)[END_REF]. The proteomic dataset obtained here represents 54%

(including 15% of membrane proteins) of the predicted protein-coding genes found in the draft genome of Microbacterium oleivorans A9 [START_REF] Ortet | Draft genome sequence of Microbacterium oleivorans strain A9, a bacterium isolated from Chernobyl radionuclide-contaminated soil[END_REF]. The distribution of the peptides detected during our experiment along the chimeric genome of Microbacterium oleivorans A9 is uniform and confirm the validity of this proteogenomics approach, as previously discussed for the proteogenomics mapping of the alphaproteobacterium Tistlia consotensis [START_REF] Rubiano-Labrador | Proteogenomic insights into salt tolerance by a halotolerant alpha-proteobacterium isolated from an Andean saline spring[END_REF].

Functional categorization of the detected proteins and relative abundances

The dataset of 1,532 identified proteins were classified into 22 COG categories. Table 1 shows the functional classification of the mass-spectrometry certified global proteome. Most of the detected proteins (50%) are distributed in five classes: hypothetical proteins (15%), amino acid metabolism (11%), carbohydrate metabolism (9%), translation, ribosomal structure, and biogenesis (9%) and transcription (6%). The remaining 17 classes represent less than 5% each (Table 1). The prevalence of the same classes has been shown in previous studies [START_REF] Orellana | Proteome of Geobacter sulfurreducens in the presence of U(VI)[END_REF][START_REF] Yung | Shotgun proteomic analysis unveils survival and detoxification strategies by Caulobacter crescentus during exposure to uranium, chromium, and cadmium[END_REF], but here, the large number of uncharacterized proteins is indicative of an important lack of knowledge regarding soil gram-positive bacteria. 36 proteins account for 25% of the total proteome detected, and thus are the key molecular draft horses within the cells. These 36 most abundant proteins detected throughout the study as evaluated with their normalized spectral abundance factor (NSAF) are given in Supplementary data Table S2. The most abundant protein is the HU bacterial nucleoid DNA-binding protein involved in DNA structuration. Two of the top five most abundant proteins are the chaperonin GroEL (1.28%) and the co-chaperonin GroES (1.06%). These two proteins form the binary GroEL/GroES complex, which mediates proper folding of many proteins. The sub-unit GroEL is specifically known for its important role both in normal and stressful conditions as well as in the resistance towards toxic metals [START_REF] Weissman | Characterization of the active intermediate of a GroEL-GroES-mediated protein folding reaction[END_REF]. As expected the ribosomal proteins are abundant since the cells were collected during the exponential growth phase. The 100 most abundant proteins represent 46.1% of total NSAF. Amongst these, 16 proteins (8.3% of total NSAF) are components of the small ribosomal subunit and 27 proteins (10.6% total NSAF) belong to the large ribosomal subunit. This abundance of ribosomal proteins has already been observed [START_REF] Zhu | Tissue-specific proteogenomic analysis of Plutella xylostella larval midgut using a multialgorithm pipeline[END_REF][START_REF] Christie-Oleza | Proteomic insights into the lifestyle of an environmentally relevant marine bacterium[END_REF]. Within the most abundant proteins, several are involved in energy metabolism such as the F0F1-type ATP synthase (0.78% and 0.57% respectively).

Proteome dynamics upon uranium stress

Common differentially produced proteins over the time course of the experiment

Proteins with significant abundance changes (fold change ≥1.5 and p-value ≤ 0.05) in uraniumexposed condition versus control were listed at each time point. A total of 592 proteins met the criteria: 391 at 0.5h, 294 at 4h and 122 at 24h. The decrease of differentially produced proteins over time is due to the non-growing exposure conditions. Figure 2 shows a Venn diagram representation where the numbers of these proteins are reported for each condition, highlighting the proteins specific of a given time point and those common in two or three time points. A relatively small set of 24 proteins were shared along the whole kinetic. Remarkably seven of these 24 proteins are predicted to be ABC-type transporters. Their spectrum activities appeared to be broad as they share significant sequence similarities with dipeptide/oligopeptide/nickel, Fe 3+ -hydroxamate, multidrug or enterocholin transporters. A serine protease and a DNA-binding transcriptional regulator belonging to the PadR family are also found more abundant in presence of uranium for the three time points:

1.7, 1.6 and 4.6-fold; 2.1-fold, 2.6-fold and 2.0-fold, respectively. Members of the PadR family regulate different pathways such as multidrug resistance and detoxification [START_REF] Fibriansah | Crystal structures of two transcriptional regulators from Bacillus cereus define the conserved structural features of a PadR subfamily[END_REF].

A total of 128 proteins were found significantly more abundant in 0.5h and 4h samples of exposure to uranium compared to controls. These 128 proteins covered a large variety of molecular functions making difficult to draw specific uranium responsive pathways. Six ABC-type transporters were observed among this large panel of up-regulated proteins. They are predicted to be involved in amino acid, sugar, cobalamin, Fe 3+ siderophore and glycerol-3-phosphate transport. These data reinforce the hypothesis that bacteria deploy a rather large panel of transport machineries to respond to uranium exposure. Four proteins involved in cell division are also significantly more abundant at these two initial phases of uranium exposure: the cell division proteins FtsI and FtsQ, and two chromosome partitioning ATPases. Such observation reinforces the idea that cells are viable and active.

Once again, a link with iron metabolism is highlighted in the proteins common after 0.5h and 24h of uranium exposure with the presence of ABC-type Fe 3+ /spermidine/putrescine transport system (2.0 and 1.8-fold), the ferredoxin-NADP reductase (1.9 and 1.9-fold), the deferrochelatase/peroxidase EfeB (1.7 and 1.8-fold) and the NADPH-dependent ferric siderophore reductase (1.8 and 1.6-fold).

Interestingly, a protein predicted to be a DNA-binding transcriptional regulator belonging to the ArsR family is found significantly more abundant at 0.5h but less abundant after 24h uranium exposure.

This regulator is known to repress expression of stress-induced operons involved in toxic metal ion tolerance [START_REF] Silver | A bacterial view of the periodic table: genes and proteins for toxic inorganic ions[END_REF]. This suggests that Microbacterium oleivorans A9 first represses these genes when cells are at the initial stage of uranium contact, but need the expression of these genes when the uranium is intracellularly biomineralized and so less bioavailable.

A comparison of the significant changes of protein abundances between 4h and 24h of exposure shows that 17 proteins are in common for these two time points. Three are related to osmoregulation (DNA-binding response regulator of the OmpR family and of the AcrR family, maltooligosyltrehalose synthase) which is consistent with the exposure condition used i.e. NaCl 0.1M.

Proteins specifically modulated at a given time point

A large set of proteins (391) has been found significantly modulated in terms of abundance after 0.5h exposure to U(VI) as compared to the control. Table 2 shows the proteins with the most important changes (fold-change ≥ 3.5). The chorismate mutase exhibited the highest fold-change (5.8-fold), while its homologue from Geobacter sulfureducens has been previously shown to decrease in response to uranium [START_REF] Orellana | Proteome of Geobacter sulfurreducens in the presence of U(VI)[END_REF]. This protein is involved in phenylalanine and tyrosine synthetic pathway through the conversion of chorismate in prephenate. As reported in Figure 3, five other proteins from the same pathway have an increased abundance upon uranium exposure, including the isochorismate synthase (2.6-fold) and the aminotransferase/4-amino-4-deoxychorismate lyase (2.0fold). A protein involved in ABC-type glycerol-3-phosphate transport, two proteins responsible of acyl transfer to glycerol-3-phosphate and the CDP-glycerol glycerophosphotransferase were more abundant in uranium condition. Glycerol-3-phosphate appears as an important metabolite which may provide phosphate for complexing and immobilizing uranium [START_REF] Martinez | Aerobic uranium (VI) bioprecipitation by metal-resistant bacteria isolated from radionuclide-and metal-contaminated subsurface soils[END_REF]. Major changes affected the phosphate metabolism as evidenced by the important number [START_REF] Kulkarni | Interaction of Uranium with Bacterial Cell Surfaces: Inferences from Phosphatase-Mediated Uranium Precipitation[END_REF] of proteins related to this metabolism. A large panel of phosphatases (16 polypeptides) is found in higher amounts after uranium stress. These enzymes are involved in phosphate removal mainly from sugar (fructose, trehalose) and from amino acids (serine, threonine).

Regarding the two other time points analyzed by high-throughput proteomics, the most modulated protein detected after 4h is also the protein with the higher abundance after 24h of uranium exposure compared to control (11-fold and 25-fold respectively). This protein shares some sequence similarities (63% identity) to the uncharacterized YkoI membrane protein of Microbacterium oleivorans LKL04 (Table 2). It presents a PepSY domain (pfam 03413) hypothetically involved in inhibition of a peptidase activity. Based on the TMHMM predictive tool [START_REF] Krogh | Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes[END_REF], one transmembrane segment has been identified in the Microbacterium oleivorans A9 protein homolog from residues 39 to 61, suggesting that the longest part of this protein is extracellular (residues 62 to 247). Regarding the synteny in Microbacterium oleivorans A9 genome, a signal transduction histidine kinase is adjacent to this protein, indicating a possible involvement in signaling function. One of the four proteins for which the abundance decreased at 4h of uranium exposure is the glutaredoxin which can interact physically with a mercury/uranyl reductase (MerA) and plays a role in toxic metals resistance [START_REF] Cassier-Chauvat | Responses to oxidative and heavy metal stresses in cyanobacteria: recent advances[END_REF]. After 24h exposure, 12 transporters involved in the transport of diverse compounds (lipoprotein, Fe 3+ -hydroxamate, dipeptide/oligopeptide/nickel, enterochelin, Fe 3+ /spermidine/putrescine) are more abundant in the uranium condition compared to the control (Table 2). A DNA-binding response regulator of the NarL/FixJ family is also more detected (5.3-fold).

The homologous regulator from Escherichia coli has been characterized and found to control nitrateand nitrite-regulated gene expression. This is concordant with the lower detection (-2.1-fold) of a ferredoxin subunit of the nitrite reductase. In the present exposure condition, nitrate has been added in both control and uranium conditions and probably contributes to modify the production of these two proteins.

Phosphate metabolism

In our previous study, we have evidenced a phosphate efflux concomitant to the uranium efflux between 0.5h and 4h exposure and the intracellular formation of autunite, a mineral made of U, P and Ca, suggesting a strong connection between phosphate and uranium [START_REF] Theodorakopoulos | Use of combined microscopic and spectroscopic techniques to reveal 20 interactions between uranium and Microbacterium sp. A9, a strain isolated from the Chernobyl exclusion zone[END_REF]. Among these proteins, the phosphohistidine phosphatase SixA is the most up-produced protein (3.5 fold-change at 0.5h and 4h). Its precise role is still unclear but its involvement in a signal transduction circuitry through down-regulation of the ArcB-to-ArcA phosphorelay has been shown under anaerobic conditions in Escherichia coli [START_REF] Matsubara | The SixA phospho-histidine phosphatase modulates the ArcB phosphorelay signal transduction in Escherichia coli[END_REF]. On the other hand, the increase abundance of the broad specificity phosphatase PhoE only after 4h (2.3 fold-change) is congruent with the phosphate efflux observed in our previous experiment [START_REF] Theodorakopoulos | Use of combined microscopic and spectroscopic techniques to reveal 20 interactions between uranium and Microbacterium sp. A9, a strain isolated from the Chernobyl exclusion zone[END_REF]. Several components of an ABC-type glycerol-3-phosphate transport system have also a significant positive fold-change. No such component is found more abundant after 24h exposure probably because the phosphate is complexed with uranium leading to autunite accumulation. The glycerol-3-phosphate has been demonstrated as a source of phosphate for the precipitation of uranium [START_REF] Martinez | Aerobic uranium (VI) bioprecipitation by metal-resistant bacteria isolated from radionuclide-and metal-contaminated subsurface soils[END_REF]. A polyphosphate kinase shows an up-production after 0.5 h (foldchange of 1.8). It can act as a defense mechanism by constituting a phosphate reserve that will be used latter to form uranyl-phosphate complex. As reported in Figure 4, the genomic context of this kinase reveals a locus with seven adjacent genes, possibly organized as an operon. These genes are all related to phosphate metabolism. The operon comprises a transcriptional regulatory protein, the phosphohistidine phosphatase SixA and four components of an ABC-type phosphate transport system. Five of this six genes encode proteins which have been found more abundant in uranium condition but with low statistical confidence (p-value above 0.05) or with significant but low foldchange. Noteworthy, this cluster of genes is conserved amongst several Microbacterium species from our collection (data not shown). In this work, several proteins involved in phosphate metabolism were modulated upon uranium exposure. The relationships between uranium and phosphate metabolism have also been evidenced by others [START_REF] Macaskie | Microbially-enhanced chemisorption of heavy metals: a method for the bioremediation of solutions containing long-lived isotopes of neptunium and plutonium[END_REF][START_REF] Beazley | Uranium biomineralization as a result of bacterial phosphatase activity: Insights from bacterial isolates from a contaminated subsurface[END_REF][START_REF] Kulkarni | Interaction of Uranium with Bacterial Cell Surfaces: Inferences from Phosphatase-Mediated Uranium Precipitation[END_REF].

Reminding that uranium can bind to highly oxygenated sites such as phosphate and carbonate, it is interesting to point out that a component of an ABC-type nitrate/sulfonate/bicarbonate transport system shows a significant increase in terms of abundance (1.9 fold-change) after an exposure of 4h, in the same time as uranium and phosphate efflux occur. Several cations transporters (K + , Mn 2+ , Zn 2+ , Mg 2+ , Co 2+ ) are also induced upon uranium exposure. One can speculate that one of these transporters may be involved in uranyl efflux.

Iron metabolism

In our previous study and unlike to phosphate metabolism, we obtained no element suggesting an effect of uranium on iron metabolism [START_REF] Theodorakopoulos | Use of combined microscopic and spectroscopic techniques to reveal 20 interactions between uranium and Microbacterium sp. A9, a strain isolated from the Chernobyl exclusion zone[END_REF]. Here, however, a strong impact on iron metabolism is highlighted through the large number of proteins related to iron metabolism more abundant under uranyl stress (19 out of 43 proteins classified in the "Inorganic ion metabolism and transport" category). Most of them are components of the siderophore iron uptake system, either ABCtransport type subunits or siderophore modification enzymes. Among these 19 proteins, two ABC-type Fe 3+ -hydroxamate transport systems and one ABC-type enterocholin transport system are found systematically more abundant at the three time points (fold changes of 1.7, 2.1 and 2.8 respectively).

Hydroxamate and enterocholin are siderophores, low molecular compounds produced by bacteria to scavenge iron (Fe(III)) in iron starvation conditions. The involvement of siderophores in tolerance to toxic metals has already been demonstrated [START_REF] Rajkumar | Potential of siderophore-producing bacteria for improving heavy metal phytoextraction[END_REF]. The ATPase component of an ABC-type cobalamin/Fe 3+ -siderophore transport system is also found in higher proportion at 0.5h and 4h with fold changes of 1.6 and 1.7 respectively. Moreover, the abundance of a NADPH-dependent ferric siderophore reductase increased upon uranium exposure: fold change of a 1.8 and 1.6 at 0.5h and 24h, respectively. This protein is involved in the release of iron from siderophore. Involvement of siderophores in the resistance to uranium has been poorly studied, but hydroxamate has been shown to bind uranium and to be able to chelate more efficiently uranium with the carbonate form [START_REF] Mo | Production and characterization of desmalonichrome relative binding affinity for uranyl ions in relation to other siderophores[END_REF]. In addition, induction of siderophore production in response to uranium stress has been shown

for the marine cyanobacterium Synechococcus elongatus BDU 130911 [START_REF] Rashmi | Siderophore mediated uranium sequestration by marine cyanobacterium Synechococcus elongatus BDU 130911[END_REF]. Taken together, these data suggest that uranium could interact with siderophore in Microbacterium oleivorans A9. One attractive hypothesis is that uranium could then enter the cells via this pathway.

Two components (EfeO and EfeB) of the EfeUOB transporter were also detected in higher amounts in our dataset. This transporter is involved in iron uptake in Bacillus subtilis [START_REF] Miethke | The Bacillus subtilis EfeUOB transporter is essential for high-affinity acquisition of ferrous and ferric iron[END_REF]. EfeB is a peroxidase involved in oxidation of ferrous iron to ferric iron which allows its binding by the EfeO component.

This protein shows a regular increase at 0.5h, 4h, and 24h with fold changes of 1.7, 1.8 and 1.8, respectively. Moreover, EfeB presents the ability to reduce the reactive oxygen species formed in presence of ferrous iron and thus, confers protection to the cell. The prediction of both a cupredoxinlike domain and an imelysin peptidase with a highly conserved HXXE motif confirm that we are dealing with the EfeO component and not the EfeM for which the cupredoxin-like domain is absent [START_REF] Rajasekaran | Isolation and characterisation of EfeM, a periplasmic component of the putative EfeUOBM iron transporter of Pseudomonas syringae pv. syringae[END_REF]. The EfeO component is a ferric iron binding protein and is able to transfer its substrate to the permease EfeU. The variation in the abundance of EfeO during the experiment is not significant. The EfeU component remained undetected despite its prediction in the genome. The EfeUOB transporter of Escherichia coli has been shown to be induced under low pH condition [START_REF] Cao | EfeUOB (YcdNOB) is a tripartite, acid-induced and CpxAR-regulated, low-pH Fe2+ transporter that is cryptic in Escherichia coli K-12 but functional in E. coli O157:H7[END_REF]. This three-component transporter is also found in Bacillus subtilis and was shown to be involved in high-affinity uptake of both ferrous (Fe(II)) and ferric iron (Fe(III)) [START_REF] Miethke | The Bacillus subtilis EfeUOB transporter is essential for high-affinity acquisition of ferrous and ferric iron[END_REF]. Taken together, these results suggest that uranium exposure is perceived by Microbacterium oleivorans A9 as an iron starvation, thus enhancing the synthesis of iron uptake systems. This result is in line with what has been shown for Geobacter sulfurreducens in which uranium induces the Fur operon [START_REF] Orellana | Proteome of Geobacter sulfurreducens in the presence of U(VI)[END_REF]. Other biological molecules involved in iron metabolism, e.g. transferrin, have been shown to be able to link uranium and can constitute a way for uranium to be transported in the cells [START_REF] Hemadi | Can uranium be transported by the ironacquisition pathway? Ur uptake by transferrin[END_REF].

Concluding remarks

The high-throughput proteogenomic methodology applied to Microbacterium oleivorans A9 allows to quickly identify a large number of proteins and highlights those that could be involved in uranium tolerance. The proteome coverage obtained here is higher than previous proteomic studies devoted at exploring the proteome under uranium stress [START_REF] Orellana | Proteome of Geobacter sulfurreducens in the presence of U(VI)[END_REF][START_REF] Yung | Shotgun proteomic analysis unveils survival and detoxification strategies by Caulobacter crescentus during exposure to uranium, chromium, and cadmium[END_REF]. Microbacterium oleivorans A9 modifies drastically its proteome upon uranium exposure. As previously hypothesized, proteins involved in phosphate metabolism are found in larger abundances under uranium stress due to higher synthesis or poorer degradation or both of these mechanisms. More surprisingly, proteins related to iron metabolism were detected with significant changes in abundance. The detailed relationships between uranium response and iron metabolism are unknown at the moment. However, based on our current results it can be hypothesized that uranium may enter the cells via siderophore transportation. This will be further investigated. The detection of 15% of functionally unassigned proteins highlights the lack of knowledge regarding proteins with potentially key roles in response to uranium stress. Focusing on the most interesting protein candidates could be done by a comparative analysis using high-throughput proteogenomics between Microbacterium oleivorans A9 and other Microbacterium isolates differing in uranium sensitivity such as Microbacterium lemovicicum ViU22

[60] which is uranium-sensitive.

  established. The total spectral count of each polypeptide was used to rank the proteins from the highest to the lowest detection intensities. The statistical protein variation among the four replicates samples of the two specific exposure conditions compared was calculated using the T-Fold option of the PatternLab 2.0 software[START_REF] Carvalho | Identifying differences in protein expression levels by spectral counting and feature selection[END_REF]. This module allows normalizing the spectral count datasets, calculating the average fold changes with statistics (t-test), and estimating the resulting theoretical false discovery rate. MS/MS data were compiled in Excel (Microsoft) and converted for PatternLab with an Excel home-designed macro. Normalization was done taking into account the total number of spectral count for each sample, taking at least two readings per protein. A minimum value of 1 was added systematically to all spectral count values in order to consider missing values as a standard PatternLab normalization. Parameters for the comparisons were as follows: minimum fold change of 1.5, minimum p-value of 0.05 and BH-FDR Alfa of 0.15. Normalized spectral abundance factor (NSAF)

Figure 1 -

 1 Figure 1 -Shotgun nanoLC-MS/MS strategy used for the proteome analysis of Microbacterium

Figure 2 -

 2 Figure 2 -Venn diagram showing the number of proteins with significant abundance changes in

Figure 3 -

 3 Figure 3 -Example of the phenylalanine, tyrosine and tryptophan biosynthesis pathways. The

Table 1 . Functional classification of the proteins detected by at least two peptides. 618

 1

	COG category	Relative	Number of
		abundance (%)	proteins
	Hypothetical protein	14,6	223
	Amino acid transport and metabolism	11,0	168
	Translation, ribosomal structure and biogenesis	9,3	142
	Carbohydrate transport and metabolism	8,8	135
	Transcription	6,2
	Energy production and conversion	5,5
	Coenzyme transport and metabolism	5,3
	General function	5,1
	Lipid transport and metabolism	4,4
	Nucleotide transport and metabolism	4,3
	Posttranslational modification, protein turnover, chaperones	4,0
	Cell wall/membrane/envelope biogenesis	3,9
	Replication, recombination and repair	3,9
	Signal transduction mechanisms	3,6
	Function unknown	2,8
	Inorganic ion transport and metabolism	2,8
	Defense mechanism	1,3
	Cell cycle control, cell division, chromosome partitioning	1,1
	Secondary metabolites metabolism	1,0
	Cell motility	0,5	8
	Intracellular trafficking, secretion, and vesicular transport	0,5	8
	RNA processing and modification	0,1	1
	619

Table 2 -Key proteins with major changes after uranium exposure 620 ORFeome ID a Functional annotation Functional classification

 2

	c
	Best hit
	p value
		b
	Fold	change

a

ORFeome ID refers to Supplementary Data Table

S2

. b Fold changes represent the expression with uranium exposure normalized to expression without uranium. c The best hit correspond to the first hit obtained by BLASTp against Swissprot database.

Acknowledgments

Nicolas Gallois was financially supported by the Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA) by means of the program "Thèse Amont-Aval" directed by Yves Bréchet, Haut-Commissaire à l'Energie Atomique. The project was supported by CEA through the Toxicology program (BEnUr). We thank Microb&co for all the writing advices given during the 7 th ICME .

List of supplementary material

Supplementary Table S1. List of proteins and their spectral count in A9 samples in the first proteogenomic search. S2. List

Supplementary Table


Lucie Laurent

Caroline Lemaitre

Anne Minello

Aurélie Plessier

Géraldine Lamblin

Armelle Poujol-Robert

Anne Gervais-Hasenknopf

Emile-Alexandre Pariente

Pauline Belenotti

Nassim Mostefa-Kara

E.-A Pariente

P Sogni

M Legrand

J.-M Cournac

F Tamion

G Savoye

P Bedossa

D.-C Valla

V Vilgrain

O Goria

Cholangiopathy in critically ill patients surviving beyond the intensive care period: a multicentre survey in liver units

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

| INTRODUCTION

Cholangiopathy in critically ill patients, so-called intensive care unit (ICU) cholangiopathy, is a recently described ischaemic-like form of cholangiopathy. The first case was reported in 2001 by Scheppach et al. [START_REF] Scheppach | Sclerosing cholangitis and liver cirrhosis after extrabiliary infections: report on three cases[END_REF] Reports of isolated cases or small series have mostly originated from intensive care units, in patients with polytrauma, infection, burn, major surgery or respiratory failure. [START_REF] Scheppach | Sclerosing cholangitis and liver cirrhosis after extrabiliary infections: report on three cases[END_REF][START_REF] Lin | Sclerosing cholangitis in critically ill patients: an important and easily ignored problem based on a German experience[END_REF][START_REF] Gelbmann | Ischemic-like cholangiopathy with secondary sclerosing cholangitis in critically ill patients[END_REF] ICU cholangiopathy is characterised by the formation of biliary casts at an early stage.

Subsequent changes include the progressive destruction of intrahepatic bile ducts with multifocal strictures and dilatations. [START_REF] Lin | Sclerosing cholangitis in critically ill patients: an important and easily ignored problem based on a German experience[END_REF][START_REF] Gelbmann | Ischemic-like cholangiopathy with secondary sclerosing cholangitis in critically ill patients[END_REF][START_REF] Al-Benna | Secondary sclerosing cholangitis, following major burn injury[END_REF] Rapid progression to acute liver failure has been reported. [START_REF] Gelbmann | Ischemic-like cholangiopathy with secondary sclerosing cholangitis in critically ill patients[END_REF] Average survival time, without liver transplantation, is only 13 months. [START_REF] Kulaksiz | Poor outcome in progressive sclerosing cholangitis after septic shock[END_REF][START_REF] Jaeger | Secondary sclerosing cholangitis after long-term treatment in an intensive care unit: clinical presentation, endoscopic findings, treatment, and follow-up[END_REF][START_REF] Kirchner | Outcome of patients with ischemic-like cholangiopathy with secondary sclerosing cholangitis after liver transplantation[END_REF] Pathogenesis has been poorly investigated. [START_REF] Gelbmann | Ischemic-like cholangiopathy with secondary sclerosing cholangitis in critically ill patients[END_REF][START_REF] Ruemmele | Secondary sclerosing cholangitis[END_REF] Currently available clinical surveys have been conducted in ICU and are mostly based on endoscopic retrograde cholangiopancreatography (ERCP) findings, providing little information on the sequelae of the disease in patients surviving intensive care and thus referred to liver units. Therefore, the aims of this present survey were to describe the clinical, laboratory and imaging (MRI) features, as well as the characteristics and the outcome of patients with ICU cholangiopathy referred to liver units.

| PATIENTS AND METHODS

Cases were retrospectively identified through a survey conducted in French liver units affiliated with two French reference centres for rare diseases: the vascular liver diseases centre at Beaujon University Hospital, in the greater Paris area; and the inflammatory biliary diseases centre at Saint Antoine University Hospital, Paris, France.

Diagnosis of cholangiopathy in critically ill patients was based on the following three criteria: (1) normal liver function tests before admission to ICU; (2) development of persistent cholestasis after admission to ICU; and (3) irregularities of the intrahepatic ducts on magnetic resonance cholangiopancreatography. Patients were excluded from the study if they had a history of liver disease or inflammatory bowel disease prior to ICU admission; and if trauma had induced injury to splanchnic vessels. Cholestasis was defined as an increase in alkaline phosphatase (AP) activity more than twice the upper limit of normal values (ULN). Clinical, laboratory, histopathological and imaging data were collected using a structured questionnaire, on the day of admission, during ICU stay and during post-ICU follow-up. ICU records were analysed for cause of admission, use of mechanical ventilation, vasopressors, transfusions, systemic inflammatory response syndrome (SIRS), sepsis and antibiotic therapy.

One expert abdominal radiologist (V.V.) reviewed all MRI while blinded to clinical and laboratory parameters. The following sequences were analysed: (1) T1-weighted sequence, axial, (2) fatsuppressed T2-weighted sequence, axial, (3) single-shot T2-weighted sequence, axial HR, (4) diffusion-weighted sequence, (5) 2D and 3D MR cholangiography, (6) contrast-enhanced sequences using extracellular contrast agents. All sequences including native and 3D MIP (Maximal intensity projection) reconstructions were analysed on a workstation. For each patient, the following items were studied using a standardised scale: intrahepatic biliary duct abnormalities, main bile duct abnormalities, abnormalities of the hepatic parenchyma, parenchymal enhancement after injection, enhancement of bile duct walls, hepatic arterial abnormalities, signs of portal hypertension (splenomegaly, portosystemic collaterals) and morphological changes of the liver.

Treatment modalities during follow-up were collected including: endoscopic treatment, ursodeoxycholic acid therapy and liver transplantation.

| Statistical analysis

All results are expressed as median and range. Categorical data are expressed as frequency and percentage.

| RESULTS

We identified 19 patients with cholangiopathy, 3 of whom were excluded from analysis because of mesenteric deinsertion in one, hepatic artery dissection and inability to review the images in the others.

Finally, 16 patients were included (11 male, 5 female). The median age at diagnosis was 52 years (16-83). Seven patients had at least one cardiovascular risk factors on admission (4 patients were current smokers, 3 had hypercholesterolemia, 1 arterial hypertension, 1 diabetes). None of the patients reported alcohol misuse. One patient had received chemotherapy for B-cell lymphoma. One patient had a documented MDR3 (ABCB4) mutation. As shown in Table 1, extensive burns and polytrauma were the predominant reasons for ICU admission. All patients presented with hemodynamic instability on admission. Eleven patients required noradrenaline treatment. The median duration of noradrenaline therapy was 6 days (1-42). Fifteen patients required transfusions. The median number of red blood cell units (RBC) transfused was 26 (range: 2-94). Fifteen patients had mechanical ventilation for a median duration of 51 days (range: 20-136).

The median duration of ICU stay was 72 days (range: 1-172). As presented in Table 2, the increase in serum AP levels started after a median time of 11 days, reaching a peak of 15 on average and at the upper limit of normal values (ULN), a median of 3 months after admission. However, there were marked variations in time course according to patients. During hospitalisation, all patients presented a systemic inflammatory response syndrome (SIRS) and at least one bacterial infection. The most frequent infections were septicaemia in 13 patients, pneumonia in 13 and skin infection in 12. Infections were caused by various species (Pseudomonas aeruginosa, enterobacter cloacae, streptococcus pneumoniae, staphylococcus aureus etc.). Bacterial examination of the bile was not performed.

All patients had at least one MRI examination of the liver including MR cholangiography (range, 1-3). Median delay between ICU admission and first MRI was 4 months (0.5-20.0). At initial MRI, 11 of 16 patients had endoluminal defects of the bile ducts corresponding to biliary casts, which was confirmed by ERCP in three patients (median: 3.5 months [0.5-7.6]). Five patients had biliary leakage with hyperintense collections on T2-weighted MR images communicating with bile ducts, suggestive of bilomas (median: 3.8 months [0.5-6.5]) (Figure 1). All but three patients had multiple strictures of the intrahepatic bile ducts. Strictures were >1 cm in length at the level of the right, left or upper common hepatic ducts in 11 patients. These long strictures were associated with shorter strictures of peripheral or peripheral and central intrahepatic bile ducts. Fifteen patients had thickened and enhancing intrahepatic bile duct walls (Figure 2). The common bile duct (CBD) was not dilated and there was no abnormal thickening of the CBD wall. Liver morphology was normal in all but two patients at the first MRI (hypertrophy of the caudate lobe and segment IV for one and hypertrophy of segment IV for the other).

The hepatic artery and its main branches were patent in all patients on contrast-enhanced MR images. Three patients were lost to follow-up after a median delay of 5.3 months (4.9-30.9), leaving 13 patients for outcome analysis.

Median follow-up was 20.6 months (range: 4.7-71.8) (Table 3). All

| DISCUSSION

We report here, a retrospective multicentre study on a cohort of 16 patients surviving a critical illness in ICU and affected with cholangiopathy, as diagnosed subsequently by hepatologists. Although the small number of patients and the heterogeneous management, all related to the rarity of the disease, constitute limitations to the A major strength of the present study was the availability of both initial MRI and MR cholangiography imaging, as well as followup MRI in half of the cohort. In previously reported surveys with two exceptions, [START_REF] Kwon | Biliary cast formation with sclerosing cholangitis in critically ill patient: case report and literature review[END_REF][START_REF] Benninger | Sclerosing cholangitis following severe trauma: description of a remarkable disease entity with emphasis on possible pathophysiologic mechanisms[END_REF] imaging studies were limited to ERCP. Conversely, in the current study we observed different imaging patterns according to the delay of the disease. At an early stage (median:

T A B L E 1 Length
3.5 months), imaging showed luminal alterations of the intrahepatic biliary tree corresponding to biliary casts and biliary leakages in most patients. At a later stage, MR cholangiography showed long hilar strictures, whereas the distal common bile duct appeared normal.

The findings of our multicentre survey in liver units independently validate previously reported observations in ICU based surveys; notably a mean age of 50 years, a male predominance, and a rapid development of cholestasis after admission to ICU in patients who had no prior history of liver disease and normal liver function tests before admission. [START_REF] Lin | Sclerosing cholangitis in critically ill patients: an important and easily ignored problem based on a German experience[END_REF][START_REF] Gelbmann | Ischemic-like cholangiopathy with secondary sclerosing cholangitis in critically ill patients[END_REF][START_REF] Al-Benna | Secondary sclerosing cholangitis, following major burn injury[END_REF] Cholestatic liver function tests are very common in ICU, due to sepsis, whatever the origin, or due to parenteral feeding or are druginduced, however, the vast majority of such cases do not have demonstrable cholangiopathy. MRI is indicated when cholestasis persists after recovery from the ICU stay and with particularly elevated levels of alkaline phosphatase or when clinical features suggest biliary complications such as cholangitis.

A second strength of this present survey was the availability of long-term follow-up data in a specific subset of patients having survived ICU stay, a population of major interest for hepatologists. The outcome of cholangiopathy seems better than that reported in the literature, as described in Table 4. Indeed, most published cases have been reported by intensivists, who diagnosed severe cholangiopathy, cholangitis, liver abcesses and poor outcome without transplantation during ICU stay. Kulaksiz et al in 2008 estimated a median survival time of 13 months without liver transplantation. [START_REF] Kulaksiz | Poor outcome in progressive sclerosing cholangitis after septic shock[END_REF] After a median follow-up of 20.6 months, 11 of our patients (68.8%) were alive without liver transplantation, including 9 with cholestasis but without jaundice. However, two patients died due to acute liver failure, pointing to an extreme variability in the expression and outcome of the disease. These two patients presented to ICU with early severe disease as described in previously reported surveys. The design of our study does not allow for the identification of prognostic factors for the severity of bile duct disease.

In our study, most patients had burns and trauma (11 of 16 patients and 3 of 16 patients respectively). It must be emphasised that cases were collected on the basis of those patients who survived ICU stay, that is, patients in very good condition at the time of their critical illness. This in turn explains the predominance of accidents, (including burns and trauma) in previously healthy people as a cause for admission to ICU. Furthermore, severe burns require prolonged care and thus allowed for prolonged surveillance. Moreover, in ICU the death rate for patients with burns is lower than for all other pathologies (25% vs 50-70%), probably because these patients are younger, with fewer comorbidities and a different pathophysiology for critical conditions. [START_REF] Pham | Epidemiology and outcomes of older adults with burn injury: an analysis of the National Burn Repository[END_REF][START_REF] Higgins | Early indicators of prolonged intensive care unit stay: impact of illness severity, physician staffing, and pre-intensive care unit length of stay[END_REF] The pathophysiology of ICU cholangiopathy in critically ill patients is likely complex and multifactorial, because it appears to be rare and to affect patients with diverse underlying conditions. Unlike the hepatic parenchyma, the intrahepatic biliary tree is exclusively supplied by the hepatic arterial branches, via the peribiliary vascular plexus. [START_REF] Deltenre | Ischemic cholangiopathy[END_REF] Ischaemia due to hypovolaemia and hypoxaemia could be the first hit in cholangitis in critically ill patients, vasoconstrictive therapy might constitute an aggravating factor. [START_REF] Gelbmann | Ischemic-like cholangiopathy with secondary sclerosing cholangitis in critically ill patients[END_REF][START_REF] Weig | Abdominal obesity and prolonged prone positioning increase risk of developing sclerosing cholangitis in critically ill patients with influenza A-associated ARDS[END_REF] In our cohort, MR imaging findings showed no damage of the distal common bile duct in any of our 16 patients, which confirmed the findings of Benninger et al in 5 patients using ERCP. [START_REF] Benninger | Sclerosing cholangitis following severe trauma: description of a remarkable disease entity with emphasis on possible pathophysiologic mechanisms[END_REF] Unlike the intrahepatic biliary tree, which is exclusively supplied by the hepatic arterial branches, the common bile duct is also supplied by gastroduodenal arterial branches. [START_REF] Kramer | Mechanism and management of cholestasis in sepsis and post-operative care[END_REF] All these elements argue for the involvement of hepatic arterial microcirculation.

The clinical and imaging features of ICU cholangiopathy described here are very similar to those described in cases of ischaemic cholangiopathy after liver transplantation including bile cast formation or bilomas and the aspect of primary sclerosing cholangitis.

Several factors likely contribute to arterial ischaemia at liver

  A second MRI was performed in eight patients. Median delay between admission to ICU and second MRI was 15.6 months (5.2-27.3). Two patients developed diffuse strictures of the intrahepatic bile ducts and one had a rarefaction of the intrahepatic bile ducts. Abnormal portal veins were observed in two other patients: splenomegaly, portosystemic collaterals with thrombosis of segmental portal branches in one; and thrombosis of the left portal branch without features of portal hypertension in the other. At an early stage, most patients had defects in the intrahepatic biliary tree corresponding to biliary casts and biliary leakages. At a later stage, MR cholangiography showed long hilar strictures, whereas the distal common bile duct appeared normal. Liver biopsy, performed in 6 patients, 130 (45-668) days after admission to ICU, showed significant alterations of interlobular bile duct epithelium such as nuclear pseudostratification, cytoplasmic clarification/vacuolisation of biliary epithelial cells or cell desquamation in the bile duct lumen. Arterioles and venules were present and patent in the portal tracts. Portal and periportal mild mixed inflammatory infiltrate was also described. Ductular reaction and bile duct infarcts close to the portal tracts were observed in two patients. No periportal or lobular necroinflammatory lesions were seen. Marked centrilobular cholestasis was detected. Two years (range 1.8-3.1) after admission, we observed portal and periportal fibrosis in 4 patients and biliary cirrhosis in 2 patients.

  patients received ursodeoxycholic acid (UDCA) (10-15 mg/kg/d). ERCP with endoscopic sphincterotomy of the papilla was performed in 4 patients with persistent jaundice, 4.1 months (2.6-5.1 months) after admission to ICU. The material removed was not analysed. Two patients had persistent severe cholestasis with jaundice and severe cholangiopathy. They died from hepatic failure. The first patient died 20.5 months after admission, being considered too sick for liver transplantation. The second patient, a 79-year-old male, died 71.8 months after initial admission for hepatic encephalopathy secondary to biliary cirrhosis. No patient was transplanted. One patient had worsening strictures of the intrahepatic bile ducts with jaundice without complication. Nine patients had persistent but minor strictures of the intrahepatic bile ducts on MR cholangiography, and persistent cholestasis without jaundice. One patient had normal liver function tests.

F I G U R E 1

 1 MR cholangiography at an early stage. Defects corresponding to casts and extrabiliary collection of bile corresponding to bilomas (Left: At 2 mo after diagnosis. Right: At 7 mo after diagnosis) F I G U R E 2 Contrast-enhanced MR obtained at the portal venous phase and magnetic resonance cholangiopancreatography (At 4 mo after diagnosis): Rail thickening corresponding to thick and enhancing bile duct wall (left). Series of strictures and dilatations (right)

  of stay, use of vasoactive agents, mechanical ventilation and transfusion according to the cause of admission to ICU

			Vasoactive agents		Mechanical ventilation	Transfusion
										RBC transfusion	Length of ICU
			Patients,	Days, median	Patients,	Days, median	Patients,	unit per patient	stay-days
	Causes	Patients	n (%)		(range)		n (%)	(range)	n (%)	(median, range)	median (range)
	Burns	11	8 (73%)	5 (1-42)		11 (100%)	48 (2-58)	11 (100%)	26 (2-94)	76 (35-172)
	Polytrauma	3	3 (100%)	23 (6-32)		3 (100%)	51 (37-52)	3 (100%)	47 (7-48)	56 (40-58)
	Infection	1	0				0	69	0	1
	ARDS	1	1		3		1	69 (1-172)	1	16	69
	RBC, red blood cell units; ARDS, acute respiratory distress syndrome.
	T A B L E 2 Liver function tests
		Time to first	Time to peak	Peak level
		increase (days),	level (days),	(UNL) (median,
		(median, range)	(median, range)	range)
	AP	11 [2-46]		81 [12-511]	15 [4-32]
	gammaGT	9 [4-19]		84 [26-476]	28 [18-119]
	Total Bilirubin	26 [10-84]		74 [17-426]	15 [3-33]
	ALAT	-		43 [20-84]	7 [6-16]

UNL, upper limit of normal values; AP, alkaline phosphatase. interpretation of our study; novel and solid findings include a specific MR cholangiographic aspect, a better outcome than previously reported, and a preponderance of burns as the cause of admission to ICU.

  Comparison between data in the literature[START_REF] Pasher | Bile duct complications after liver transplantation[END_REF] : preservation injury to the peribiliary plexus, hepatic artery thrombosis, the suppression of arterial collaterals by explantation, cytomegalovirus infection and rejection. The resemblance of ICU cholangiopathy with cholangiopathy after liver transplantation with a patent hepatic artery is even more striking: indeed, the strictures also predominantly affect the middle third of the common bile duct, with relative preservation of the distal common bile duct. These similarities reinforce the ischaemic hypothesis of the ICU cholangiopathy. CONCLUSIONS MR cholangiography is an easy, non-invasive, complete imaging method in transportable patients for the diagnosis of ICU cholangiopathy in patients surviving beyond the intensive care period. The preservation of the distal common bile duct is suggestive of this diagnosis in this context. The spectrum of severity of the disease is probably wider than previously suspected: fewer severe features and better outcomes of the disease have been observed, after ICU stay, particularly in patients with burns.

	T A B L E 3 Follow-up (months) transplantation 16,
						Liver
						function tests
						(1: cholestasis
						without jaundice/2:
		Time to	Time to	Time to	Length of	jaundice/3: Normal
	Patient	MRI 1 (mo)	MRI 2 (mo)	ERCP (mo)	follow-up (mo)	liver function tests)
	1	6.5	17.4		71.8 a
	2	4.5	9.3		33.7	1
	3	7.6	14.1		20.5 a
	4	1.5	10.0		28.8	1
	5	4.8			5.3 b
	6	0.5	27.3		30.9 b
	7	4.3			65.2	1
	8	1.9			17.2	3
	9	5.3	17.0	2.6	45.4	1
	10	3.8			4.7	1
	11	3.2	5.2		15.6	1
	12	20			20.6	1
	13	4.3			4.9 b
	14	3.3		5.1	8.1	1
	15	3.5		3.9	6.1	1
	16	12.5	23.3	4.2	24.4	2
	Median	4.3	15.6	4.1	20.6
	MRI, magnetic resonance imaging; ERCP, endoscopic retrograde cholangiopancreatography.
					Liver	Death due
		Trauma	Burns		transplantation	to hepatic
	Author	(n)	(n)	Follow-up (mo)	(n)	failure (n)
	Jaeger 6	5/10	0/10	21.0 (3-54)	1/10	0/10
	Gelbmann 3	7/26	0/26	(4-49)	7/26	7/26
	Kulaksiz 5	5/29	5/29	21.0 (1-123)	7/29	18/29
	Esposito 18	1/10	1/10	14.4 (2-92)	0/10	5/10
	Our study,	3/16	11/16	20.6 (4.7-71.8)	0/16	2/16
	2017

a Death. b Lost to follow-up. T A B L E 4 5 |

ACKNOWLEDGEMENTS

We are grateful to hepatologists from 18 liver centres in France belonging to 2 national networks (the rare vascular liver diseases network and the rare biliary diseases network) for help for recruitment of cases. All of them had answered but 8 had no case to propose. We are grateful to Nikki Sabourin-Gibbs (Rouen University Hospital) for help in editing the manuscript.

Declaration of personal and funding interests: None.

AUTHORSHIP

Guarantor of the article: LL.

Author contributions: LL, DCV, VV, OG: contributed to study conception and design. LL, OG: contributed to data acquisition. LL, DCV, VV, OG: contributed to data analysis and interpretation. LL, CL, AM, AP, GL, APR, AGH, EAP, PB, NMK, PS, ML, JMC, FT, GS, PB, DCV, VV, OG: wrote the paper. LL, DCV, VV, OG : contributed to editing.

All authors approved the final version of the manuscript.