Dr Matthieu Fonvielle
email: matthieu.fonvielle@crc.jussieu.frmichel.arthur@crc.jussieu.fr

Dr Ahmed Bouhss

Coralie Hoareau

Ms Delphine Patin

Dr Dominique Mengin-Lecreulx

Dr Laura Iannazzo

Dr Nicolas Sakkas

Affaf El Sagheer

Mélanie Tom Brown

Dominique Mengin- Lecreulx

Affaf El Sagheer

Mélanie Ethève- Quelquejeu

Dr Michel Arthur
email: michel.arthur@crc.jussieu.fr

Mélanie Ethève-Quelquejeu

Dr Afaf H El-Sagheer

Synthesis of lipid-carbohydrate-peptidyl-RNA conjugates to explore the limits imposed by the substrate specificity of cell wall enzymes on the acquisition of drug resistance

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Staphylococcus aureus. Mimics of Gly-tRNA Gly and lipid intermediate II (undecaprenyl-diphosphodisaccharide-pentapeptide) were combined in a single "bi-substrate" inhibitor (IC50 = 56 nM). The synthetic route was exploited to generate substrates and inhibitors containing D-Lac instead of D-Ala at the C-terminus of the pentapeptide stem, a modification responsible for vancomycin resistance in the enterococci. The substitution impaired recognition of peptidoglycan precursors by FmhB. The associated fitness cost may account for limited dissemination of vancomycin resistance genes in S. aureus.

Various RNA conjugates have been synthesized to develop new therapeutic strategies and to investigate basic biological processes. [1] These molecules combine an oligonucleotide with carbohydrates, peptides, small molecules, aptamers, or lipids. [1a] Carbohydrate-oligonucleotide and small molecule-oligonucleotide conjugates have been mostly used to target specific receptors or to increase uptake in target organs. [2] Aptamer-RNAs were developed to improve the combined delivery of drugs and siRNAs in multidrug-resistant cancer cells. [3] Peptidyl-RNAs have played pivotal roles in mechanistic and structural studies of ribosomal and non-ribosomal peptide synthesis. [4] Peptidyl-RNA conjugates were also developed to increase the cellular uptake of siRNA. [5] Lipid-RNA conjugates were designed to increase the lipophilicity of negatively charged oligonucleotides and to leverage lipoproteinmediated endocytosis. [2] All of these conjugates offer interesting properties and access to novel chemical space. [1a] Various types of linkers have been used to conjugate RNAs to the various partners, including amide, amine, oxyamine, oxyimine, carbamate, and triazoles, which are chemically and biologically stable. [6] Bioreversible linkers rely on disulfide bridges. [7] The conjugation is mostly performed at the 5' and 3' ends of the RNA, rather than internally, with the molecules attached to the nucleobase or to the ribose.

There are limited examples of the conjugation of a single RNA molecule with multiple partners. This type of adduct is required to investigate the biosynthesis of bacterial cell walls since its major component, the peptidoglycan, is assembled from a precursor combining a disaccharide [β-1,4-linked N-acetylglucosamine and N-acetyl-muramic acid, (GlcNAc-MurNAc)], a phospholipid carrier (undecaprenyldiphosphate), and a branched pentapeptide, such as [L-Ala-D-iGlu-L-Lys(Gly5)-D-Ala-D-Ala] in Staphylococcus aureus (Figure 1a andb). Since the pentaglycine side-chain is assembled from glycyl-tRNAs by amino acid transferases of the Fem family there is interest in developing lipid-carbohydratepeptidyl-RNA conjugates (Figure 1c) to investigate the interaction of the enzymes with their substrates.

The biological question addressed in our study concerns the acquisition of resistance to the glycopeptide antibiotic vancomycin in S. aureus by replacement of the terminal D-Ala residue by D-Lac. The substitution reduces the affinity of the drugs for peptidoglycan by 1,000 fold leading to high-level resistance (Figure 1d). Since the resistance mechanism is widespread in the enterococci but only sporadically detected in S. aureus in spite of easy horizontal gene transfer between these bacteria [8] our specific aim is to evaluate whether the D-Ala to D-Lac substitution is tolerated by the S. aureus transferase FmhB responsible for incorporation of the first residue of the penta-glycine side chain. [9] We focus on FmhB since this enzyme is essential presumably because the side-chain of peptidoglycan precursors directly participates in the peptidoglycan cross-linking reaction (Figure 1e). [9][10] To investigate the specificity of FmhB, we have synthesized D-Ala-and D-Lac-containing substrates analogues (Figure 2) and lipid-carbohydrate-peptidyl-RNA conjugates acting as inhibitors (Figure 1c). This strategy was designed to assess the impact of the D-Ala to D-Lac substitution both on the catalytic efficacy of the transfer of Gly from Gly-tRNA Gly to the peptidoglycan precursors and on the affinity of FmhB for inhibitors that mimic both substrates of the enzyme (referred to as "bi-substrates"). The target inhibitor molecules (Figure 1c) comprise soluble analogues of the peptidoglycan precursors (Lipid II) covalently-linked by a triazole to the acceptor arm of tRNA Gly . Our synthesis strategy enables to modulate the length of the RNA and lipid moieties in order to obtain ligands with suitable size and solubility for investigating enzyme activity and affinity. It also enables to selectively modify the terminal residue of the peptide (D-Ala versus D-Lac). One of the major difficulty for obtaining the desired lipid-carbohydrate-peptidyl-RNA conjugates is the identification of orthogonal reactions compatible with the various functional groups specifically present in each component of the multi-RNA conjugates. In our synthetic strategy, we overcome these problems by developing post-functionalization of RNA and peptides moieties as well as a combination of enzymatic and chemical reactions (Figure 1c). Protein (PBP) mediated cross-linking for vancomycin-susceptible and -resistant strains of Staphyloccocus aureus. [11] To generate the D-Ala-and D-Lac-ending substrates of FmhB, the soluble nucleotide precursors were extracted from vancomycin-susceptible (D-Ala 5 ) and vancomycin-resistant (D-Lac 5 ) bacteria (Figure 2).

MurG

Transfer of the phospho-MurNAc-peptide moiety to the lipid and addition of GlcNAc were obtained enzymatically. [12] For these substrates, we used commercially available heptaprenyl-phosphate instead of the natural undecaprenyl-phosphate lipid carrier (bactoprenyl). Transfer of [ 14 C]Gly from [ 14 C]Gly-tRNA Gly to the heptaprenyl-containing lipid II analogue was tested by a coupled assay involving acylation of tRNA Gly by purified glycyl-tRNA synthetase (GlyRS). The product was identified by thin layer chromatography (Supplementary information, Figure S5). This analysis revealed that substitution of D-Ala by D-Lac at the C-terminus of the lipid II analogues reduces the catalytic efficacy of FmhB by 4.6 fold (Figure 3a).

There are several limitations resulting from the insolubility of the heptaprenyl-containing substrates including uncertainty regarding the concentrations of lipid II analogues actually accessible to FmhB and time consuming solvent extractions required for purification. In order to gain access to soluble lipid II analogues, we replaced heptaprenyl-phosphate by neryl-phosphate in the synthesis procedure. The resulting neryl-containing lipid II analogues were effectively used as substrates by FmhB revealing again a reduced catalytic efficacy for D-Lac-ending substrates (7 fold; Figure 3b and Table 1). Lipid-carbohydrate-peptidyl-RNA conjugates mimicking both the Gly-tRNA Gly and lipid II (lipidcarbohydrate-peptide) substrates were synthesized in order to explore the impact of the D-Ala to D-Lac substitution on the affinity of FmhB for peptidoglycan precursors (Figure 2). The presence of the carbohydrate and lipid parts have been shown to be essential for the activity of the FmhB transferase. [13] The synthesis of the Lipid II moiety started by four enzymatic reactions for sequential addition of L-Ala, D-Glu, meso-cystine, and D-Ala-D-Ala or D-Ala-D-Lac to UDP-MurNAc by Mur synthetases (Figure 4). Meso-cystine is a structure analogue of meso-2,6-diaminopimelic acid (DAP), which is recognized by the enzyme MurE and enables post-functionalization of the peptide via formation of a dehydroalanine. [14] For this purpose, we have developed a new one-pot reaction based on reduction of meso-cystine into cysteine by a soluble phosphine followed by its conversion to dehydroalanine by 2,5-dibromohexanediamide. By this approach, a dehydroalanine residue was introduced at the 3 rd position of the stem peptide. In the following step, 1,4-addition of 1-thio butynyl to the dehydroalanine afforded two stereoisomers containing an alkyne function at the extremity of the side-chain of the 3 rd residue. The (R) diastereoisomer was purified by rpHPLC as previously described. [15] MraY and MurG were permissive for the transfer of the phospho-MurNAc-peptide moieties to heptaprenyl-phosphate and neryl-phosphate and the subsequent addition of GlcNAc, respectively, generating the corresponding alkyne-containing lipid II analogues. [12] For the RNA moiety, 2'-azido-5'-dimethoxytrityl-benzoyl adenosine was coupled to a resin for solid phase synthesis of an 18-mer mimicking the acceptor arm of tRNAGly. [4a] During solid phase synthesis, a hexaethylene glycol linker was incorporated to stabilize the hairpin duplex formed by the RNA strand. The alkyne-containing lipid II analogues were coupled to the azido-containing RNA by the Cu I -catalyzed Huisgen-Sharpless cycloaddition reaction in the presence of a metal ligand (THPTA). [15] The resulting triazole-containing lipid-carbohydrate-peptidyl-RNA conjugates were purified by denaturing polyacrylamide gel electrophoresis and by rpHPLC for heptaprenyl-and neryl-containing molecules, respectively (Supplementary Information).

Our next objective was to assess the impact of the substitution of the C-terminal D-Ala by D-Lac on the inhibition of FmhB by lipid-carbohydrate-peptidyl-RNA conjugates ("bi-substrates"). The conjugates were tested as FmhB inhibitors in the GlyRS-FmhB coupled assay (Figure 5). Substitution of D-Ala by D-Lac at the C-terminus of the peptide stems of the conjugates led to a 15-fold increase in the IC50 values (from 56 ± 6 nM to 820 ± 50 nM for neryl-containing analogues; Table 1). This difference highlights the contribution of the D-Ala-D-Ala amide bond to the binding energy to the enzyme. IC50s of 56 ± 6 nM and 5 ± 3 nM were determined for D-Ala-ending heptaprenyl-and neryl-PP-disaccharide-pentapeptide-RNA conjugates indicating that shortening of the lipid moiety (7 versus 2 prenyl groups) leads to a ca.

11-fold reduction in affinity (Supplementary Information and Table 1). This mirrors the 7-and 10-fold reduction observed with the substrates (Table 1 Total [10a,16] and enzymatic [12] synthesis of lipid II have been described previously and were mostly used to incorporate fluorescent tags. Here, we report a hybrid strategy using dehydroalanine-containing lipid analogues for post-functionalization. This approach was used to generate lipid-carbohydratepeptidyl-RNA conjugates mimicking the lipid II and Gly-tRNA Gly substrates of the FmhB transferase from S. aureus. Variability was introduced in the sequence of the peptide and in the number of isoprenyl repeats in the lipid moiety. Solid-phase synthesis of the RNA moiety offers the possibility to modulate the size and the sequence of the RNA moiety. The 1,4 addition of the thiol to dehydroalanine also offers a versatile access to bi-substrates containing linkers with various lengths and structures. Our lipidcarbohydrate-peptidyl-RNA conjugates provide a versatile tool to study the tRNA-dependent amino acyltransferases involved in peptidoglycan synthesis.

In this study, we investigated the specificity of the FmhB aminoacyl-transferase by comparing substrates and inhibitors differing by the presence of D-Ala or D-Lac at the C-terminus of the stem peptide. Testing the substrates indicated that the D-Ala to D-Lac substitution impairs the activity of FmhB.

Testing the inhibitors indicated that the substitution reduced the affinity of FmhB for the peptidoglycan precursor analogues. Structural analyses of peptidoglycan purified from vancomycin-resistant S. aureus showed that a high portion of the stem peptides were devoid of the pentaglycine side-chain. [9,11] Our data indicate that impaired activity of FmhB accounts for the presence of incomplete stem peptides lacking the pentaglycine side-chain. Such incomplete precursors are only used as acyl donors by the transpeptidases responsible for the formation of peptidoglycan cross-links (Figure 1e). Our data also indicate that the production of D-Lac-ending precursors may have a fitness cost by impairing the assembly of peptidoglycan precursors. Such a fitness cost could account for the lack of dissemination of vancomycin-resistance staphylococci in spite of the sporadic detection of this type of strain in patients treated with vancomycin. The origin of this fitness cost is not known. The presence of incomplete sidechain only in the "donor" stem of dimers suggests that impaired activity of the transpeptidases might be involved.

General information

Solvents were dried using standard methods and distilled before use. Unless otherwise specified, materials were purchased from commercial suppliers and used without further purification. Spectra were recorded on a Bruker AVANCE 400 spectrometer for 1 H (250 MHz), 13 C (63 MHz), in CDCl3. Chemical shifts (δ) are expressed in ppm relative to residual 7.24 for 1 H and 77.23 for 13 C, as internal references. Mass spectroscopy (MS) spectra were carried out on a LCQ-Deca XP-Max spectrometer in positive or negative ionization mode. Heptaprenyl-phosphate was purchased from the Institute of Biochemistry and Biophysics of the Polish Academy of Sciences, Warsaw, Poland.

Mass spectrometry analysis of RNA-containing compounds

Mass spectrometry analyses of tRNA Gly , azido-RNA, and lipid-carbohydrate-peptidyl-RNA conjugates were performed by LC-MS. Liquid chromatography (LC) was performed by anionic rpHPLC using a DNAPac RP column (4 µm; 2.1 x 50 mm; Thermo Fisher Scientific) at a flow rate of 300 µl/min with a 0 to 50% MeOH linear gradient applied from 2 to 10 min in Solution A [Solution A, triethylamine (15 mM) and 1,1,1,3,3,3-hexafluoro-2-propanol (400 mM)]. For mass spectrometry (MS), liquid chromatography was coupled to a LCQ Deca XP-Max mass spectrometer operating in the negative mode.

Enzyme production and purification

The FmhB glycyl transferase from S. aureus strain Mu50 was produced in E. coli BL21 (DE3) pREP4GroEL as a fusion protein with a C-terminal hexa-histidine tag (GSHHHHHH). FmhB was purified from a clarified lysate by metal affinity (Ni Sepharose 6 Fast flow; General Electrics) and size exclusion chromatography (DEX 75 HR 26/60; General Electrics) in 100 mM phosphate buffer (pH 7.5) containing 500 mM KCl. FmhB was concentrated (10 mg mL -1 ; Amicon Ultra, cutoff 10 kDa; Millipore) and stored at -20°C following addition of one volume of glycerol per volume of purified FmhB.

The glycyl-tRNA synthetase of S. aureus strain Mu50 was produced in E. coli BL21 (DE3) pREP4GroEL and purified in two steps by metal affinity (Ni Sepharose 6 Fast flow; General Electrics) and anion exchange chromatography (Resource Q; General Electrics), as previously described. [1] The translocase MraY from Aquifex aeolicus (for the synthesis of the lipid intermediate I) and the MurG transferase from E. coli (for the subsequent addition of GlcNAc) were purified as previously described. [2] The concentration of proteins was determined by the Bradford assay (Biorad) using BSA as a standard.

tRNA production and purification

The sequence of the tRNA Gly used in this study (5'-GCGGGAGUAGUUCAACUUUUAGAACACGUUCCUUCCCGGAACGAGGUAUAGGUGUAAAUCCUAUCUUC CGCUCCA-3') corresponds to one of the five tRNA Gly of S. aureus. [3] The matrix used for in vitro transcription was obtained by PCR using three overlapping oligonucleotides that cover the entire sequence of the tRNA and enable the introduction of the T7 promoter (underlined) (5'-TTTAATACGACTCACTATAGCGGGAGTAGTTCAACTTTTAGAACA-3', 5'-ATAGGATTTACACCTATACCTCGTTCCGGGAAGGAACGTGTTCTAAAAGTTGAACTA-3', and 5'-TGGAGCGGAAGATAGGATTTACACCTATA-3'). In vitro transcription of the resulting PCR product (100 µl) was performed at 37°C for 4 h in a total volume of 450 µl containing NTPs (4 mM each; JenaBioscience), GMP (20 mM), T7 RNA polymerase (45 µg), Triton X-100 (0.05% v/v), MgCl2 (15 mM), spermidine (2 mM), dithiothreitol (10 mM), and Tris-HCl (40 mM, pH 7.5). The reaction mixture was incubated for 20 min at 65°C, DNAse was added (30 U; RQ1 RNase-Free DNAse, Promega), and incubation was continued for 1 h at 37°C. RNA was extracted with one volume of phenol (pH 4.5), followed by one volume of phenol/chloroform (1/1 v/v), and precipitated with 5 volumes of ethanol. RNA was centrifuged at 4°C, dissolved in 100 µl of buffer A (25 mM Tris-HCl, pH 7.5; 300 mM NaCl; 5 mM MgCl2), incubated for 1 min at 85°C and cooled to room temperature for 1 h. The RNA was purified by sizeexclusion chromatography (Superdex® 75 HR 10/30, General Electrics) in buffer A. The tRNA was further purified by anion exchange chromatography (DNAPAc PA100; Dionex) with a linear gradient (25 mM to 2.5 M) of ammonium acetate (pH 8.0) containing 0.5% acetonitrile (v/v). The tRNA was lyophilized twice and dissolved in RNAse-free water (Sigma). The purity was assessed by denaturing (7 M urea) polyacrylamide gel electrophoresis (12 %) and by anion exchange chromatography as described above. The concentration of tRNA was determined by amino-acylation as described below (p10).

Semi-synthesis.

Synthesis of RNAs by solid phase synthesis. The azido-RNA helix used in this study, corresponding to the acceptor arm of S. aureus tRNA Gly described above, was obtained by solid phase synthesis as previously described. [4] The helix consisted of a 7-mer (5'-GCGGGAG-3') covalently bound to an 11-mer (5'-CUUCCGCUCCAZ-3') by a hexapolyethylene glycol linker. The ribose at the 3'-terminus of the 11mer contained a 2'-azido moiety. Production of the resin substituted by the 2'-azido nucleotide, the coupling reactions, and the final deprotection step were performed as previously described. [4] Synthesis of D-alanyl-D-lactate (Scheme S1). Synthesis of the depsipeptide D-Ala-D-Lac was performed in two steps. In the first step, Boc-D-Ala-OH (3.8 mmol, 720 mg) was dissolved in dry THF at 0°C. Carbonyldiimidazole (3.8 mmol, 617 mg) was added and the reaction was allowed to proceed for 30 min. D-Lac-OtBu (3.4 mmol, 500 mg) was dissolved in dry DCM (10 mL) and slowly added to the Boc-D-Ala acyl imidazole solution at 0°C. The mixture was incubated for 24 h at room temperature. The reaction was quenched with 10 mL of 2 M acid acetic. The organic phase was washed with 20 mL of 1 M HCl, 20 mL of 1 M NaHCO3, and 20 mL of brine. The organic extract was dried over anhydrous Na2SO4, filtered, and solvents were removed under reduced pressure. The Boc-D-Ala-D-LacOt-Bu product was recovered as a clear oil (1 g, 92% yield) and was used without further purification. In the second step, crude Boc-D-Ala-D-LacOt-Bu (3.14 mmol, 1 g) was dissolved in 10 mL of DCM, cooled at 0°C, 10 mL of TFA were slowly added, and the solution was stirred at room temperature for 1 h. DCM and TFA were removed under reduced pressure and D-Ala-D-Lac was recovered as a clear oil (329 mg, 65% yield). 1 H NMR ( 250 MHz, D2O): δ 1.58 (d, 3H, 7 Hz); 1.52 (d, 3H, 7 Hz); 4.25 (q, 1H, 7 Hz); 5.19 (q, 1H, 7 Hz). MS (ESI + ) calculated for C6H12NO4 + 162.1, found 162.1.

BocHN H 3 C OH O BocHN H 3 C O O COOt-Bu H 2 N H 3 C O O COOH i ii 92% 65 % Scheme S1. (i) CDI; D-LacOt-Bu; (ii) TFA/DCM (1/1 v/v)

Synthesis of alkyne-containing UDP-MurNAc-peptide analogues ending in D-Ala or D-Lac (Scheme S2, 6a and 6b).

Semi-synthesis of the analogues involved five steps, as previously described. [5] (i) One pot addition of L-Ala, D-Glu, meso-cystine to uridine di-phospho-N-acetyl-muramic acid (UDP-MurNAc) by synthetases MurC, MurD, and MurE, respectively. (ii) Addition of D-Ala-D-Ala or Macherey Nagel) with a linear gradient of acetonitrile (0 to 5%) in ammonium acetate (50 mM; pH 5.0) applied between 10 and 40 min at a flow rate of 1 mL min -1 . The S and R isomers eluted at 34.7 and 36.9 min for the analogues ending in D-Ala (Figure S1). The S and R isomers eluted at 35.5 and 39.7 min for the D-Lac-ending analogues. The final products, 6a and 6b, were lyophilized twice and dissolved in RNAse free water (Sigma-Aldrich). Purity of the two diastereoisomers was assessed by mass spectrometry and analytical rpHPLC with a linear gradient of acetonitrile (0 to 15%) in ammonium acetate (50 mM; pH 5.0) applied between 10 and 40 min (260 nm, 1 mL min -1 ; RT = 24.4 min and 26.8 min for the (R) diastereoisomers ending in D-Ala (6a) and D-Lac (6b), respectively. Concentrations were determined by spectrophotometry (ε260nm = 10,000 M -1 cm -1 ). Yields: 19.3% and 19.6% for 6a and 6b, respectively. Alternative one pot procedure for conversion of meso-cystine to dehydroalanine in nucleotide precursors (Scheme S3, 5a and 5b). The preceding procedure requires purification of the intermediate obtained by reduction of meso-cystine since DTT is not compatible with MSH-mediated oxidationelimination. A less effort-consuming method was therefore developed based on the one pot reaction depicted in Scheme 3. The method is based on disulfide reduction by a water soluble tris(2carboxyethyl)phosphine (TCEP) coupled to 2,5-dibromohexanediamide. Briefly, the mixture comprised meso-cystine-containing precursors ending in D-Ala or D-Lac (0.5 M), TCEP (20 mM), tris-(2,5dibromohexanediamide) (100 mM), potassium phosphate (100 mM, pH 8.0), and 20% DMF. The solution of tris-(2,5-dibromohexanediamide) was freshly prepared in DMF. Incubation was performed for 1 h at 37°C. The products of the reaction were purified in two steps. (i) Size-exclusion chromatography (SuperDexpeptide 10/300 GL, general Electrics) in water (260 nm, 1 mL min -1 ; RT = 8.1 min). (ii) rpHPLC (EC 250/22 Nucleosil 100-5 C18; Macherey Nagel) with a linear gradient of acetonitrile (0 to 15%) in ammonium acetate (50 mM; pH 5.0) applied between 10 and 40 min (260 nm, 10 mL min -1 ). RTs = 16.8 min and 18.6 min for 5a and 5b, respectively. Yields: 30% and 22% (calculated for two steps) for 5a and 5b, respectively. Purification of nucleotide precursors from bacterial extracts (1a and 1b). UDP-MurNAcpentapeptide (ending in D-Ala-D-Ala, 1a) or UDP-MurNAc-pentadepsipeptide (ending in D-Ala-D-Lac, 1b) were purified from vancomycin-susceptible E. faecalis [6] and vancomycin-resistant S. aureus, [7] as previously described. [8] Briefly, bacteria were grown at 37°C in 2 L of brain heart infusion (BHI) broth, containing 50 µg mL -1 of vancomycin to induce production of UDP-MurNAc-pentadepsipeptide 1b by the S. aureus strain. Bacteria were grown to an optical density of 0.7 at 600 nm and further incubated in the presence of bacitracin (200 µg mL -1 ) for 30 min at 37 °C. Bacteria were collected by centrifugation and ice-cold formic acid (30 mL, 1.1 M) was rapidly added to the pellet. Peptidoglycan precursors were extracted for 30 min at 4°C under gentle agitation. After centrifugation (7,000 x g; 15 min) the supernatant was filtrated, desalted on a G-25 column, and lyophilized. Peptidoglycan precursors were purified by rpHPLC with a preparative C18 column (5 µm; 22 x 250 mm; Nucleosil, Macherey-Nagel) equilibrated with ammonium formate buffer (50 mM; pH 5.0) at a flow rate of 10 mL min -1 . A linear gradient of methanol was applied from 0 to 20 % between 10 and 40 min. Fractions containing the precursors were identified by mass spectrometry and lyophilized. The precursors were dissolved in water and quantified by UV absorption at 260 nm (ε260nm = 10,000 M -1 .cm -1 ). Yield: 500 nmol per litter of bacterial culture.

Synthesis of heptaprenyl-containing lipid II analogues (Scheme S4, 2a, 2b, and 7a). Synthesis of the lipid II analogues containing a heptaprenyl moiety, instead of the undecaprenyl moiety present in the peptidoglycan precursors, was performed in a one pot enzymatic reaction containing MraY from A. aeolicus, MurG from E. coli, nucleotide precursors, and heptaprenyl-phosphate, as previously described [9] with minor modifications. Briefly, the synthesis of the lipid II analogues was performed for 18 h at 37°C in a volume of 40 µl containing Tris-HCl (50 mM; pH 8.0), heptaprenyl-phosphate ammonium salt (15 mM), UDP-MurNAc-pentapeptide (12.5 mM), UDP-MurNAc-pentadepsipeptide or the alkyne containing analogue of the former nucleotide (12.5 mM), uridine 5′-diphospho-Nacetylglucosamine (UDP-GlcNAc) sodium salt (15 mM; Sigma-Aldrich), MgCl2 (10 mM), NaCl (50 mM), N,N-dimethyldodecylamine N-oxide (LDAO; 10 mM; Sigma-Aldrich), dimethyl sulfoxide (DMSO; 2% v/v; Sigma-Aldrich), MraY (1 mg mL -1 ), and MurG (0.5 mg mL -1 ). The reagents were added to lyophilized heptaprenyl-phosphate. Cefoxitine (40 µg mL -1 ) and meropenem (40 µg mL -1 ) were also present in the reaction mixture to inhibit any contaminating D,D-carboxypeptidase activity that might have been present in the MraY or MurG preparations. After 18 h of incubation, the reaction mixture was centrifuged (12,000 x g for 10 min at room temperature), 80 µl of buffer A (20 mM ammonium carbonate containing 50% methanol (v/v) were added to the supernatant, and the lipid II analogues were purified by rpHPLC (EC 250/4.6 Nucleosil 100-5 C8; Macherey Nagel) with a linear gradient (0 to 100%) of buffer B (80% methanol, 20% isopropanol; v/v) applied between 5 min and 35 min at a flow rate of 1 mL min -1 . The lipid II analogues were detected by the absorbance at 214 nm and analyzed by mass spectrometry in the negative mode (LCQ Deca XP-Max; Thermofisher). The concentration of lipid II analogues was determined by amino acid analysis after acid hydrolysis and injection into a Hitachi L8800 amino acid analyzer equipped with a 2620 MSC-PS column. Synthesis of neryl-containing lipid II analogues used as substrates of FmhB (Scheme S4, 3a, 3b, 7b, and 7c). Soluble lipid II analogues containing a neryl group were obtained in a one pot enzymatic reaction containing MraY from A. aeolicus, MurG from E. coli, nucleotide precursors ending in D-Ala 5 or D-Lac 5 , and neryl-phosphate (Z,w) (Sigma-Aldrich), as previously described, [9] with minor modifications. Briefly, the synthesis of lipid II analogues was performed in a volume of 40 µl containing Tris-HCl (50 mM; pH 8.0), neryl-phosphate litium salt (15 mM), nucleotide precursors (12.5 mM), uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) sodium salt (15 mM; Sigma-Aldrich), MgCl2 (10 mM), NaCl (50 mM), Tween20 (5 mM), MraY (1 mg mL -1 ), MurG (0.5 mg mL -1 ). The reagents were added to lyophilized neryl-phosphate lithium salt. Cefoxitine (40 µg mL -1 ) and meropenem (40 µg mL - 1) were also present in the reaction mixture to inhibit any contaminating D,D-carboxypeptidase activity that might have been present in the MraY or MurG preparations.

After 18 h of incubation at 37°C, the reaction mixture was centrifuged (12,000 x g for 10 min at room temperature), 80 µl of buffer A (20 mM ammonium carbonate) were added to the supernatant, and the lipid II analogues were purified by rpHPLC (EC 250/4.6 Nucleosil 100-5 C8; Macherey Nagel) with a linear gradient of methanol (0 to 50%) in buffer A applied between 6.5 min and 36.5 min at a flow rate of 1 mL min -1 . The lipid II analogues, detected by the absorbance at 214 nm and identified by mass spectrometry in the negative mode (LCQ Deca XP-Max; Thermofisher), were lyophilized and dissolved in RNAse free water (Sigma-Aldrich). The concentration of lipid II analogues was determined by amino acid analysis after acid hydrolysis and injection into a Hitachi L8800 amino acid analyzer equipped with a 2620 MSC-PS column. The purity of the lipid II analogues was assessed by rpHPLC (EC 250/4.6 Nucleosil 100-5 C18; Macherey Nagel) with a linear gradient of acetonitrile (0 to 50%) in ammonium formate (10 mM; pH 5.0) applied between 6.5 and 36.5 min at a flow rate of 1 mL min -1 .

Synthesis of heptaprenyl-PP-disaccharide-peptide-RNA (8a, Scheme S5).

For the heptaprenylcontaining analogue, the Huisgen-Sharpless cycloaddition reaction was performed in a volume of 20 µl containing the lipid II analogue (110 µM), the azido-RNA helix (62 µM; figure S2b), tris(3hydroxypropyltriazolylmethyl)amine (THPTA) (3.5 mM), copper sulfate (0.5 mM), sodium ascorbate (10 mM), and sodium phosphate (100 mM; pH 8.0). The reaction was allowed to proceed at 37°C for 18 h. The peptidyl-RNA was purified by denaturing polyacrylamide gel electrophoresis. Briefly, the gel (20 x 20 x 0.1 cm), [acrylamide (12%, w/v), bis-acrylamide (4.4%, w/v), urea (8 M), tetramethylethylenediamine (TEMED, 0.04% v/v), ammonium persulfate (0.08% w/v), and TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.2)] was loaded (lane width 6 mm) with the crude cycloaddition reaction (20 µl), which was lyophilized, dissolved in 2 µl of DMF, and supplemented with 5 µl of loading buffer [50% glycerol v/v containing 0.25% (w/v) bromophenol blue]. Electrophoresis was performed for 100 min at 600 V. The gel was stained with ethidium bromide (0.5 mg mL -1 ) and imaged with a Herolab E.A.S.Y 429K camera. RNA-containing band was cut off from gel with a sterile scalpel (Figure S2a) and electroeluted in TBE buffer for 2 h at 100 V in MINI GeBaFlex-tube, 8 kDa cutoff; Gene Bio-Application, L.T.D.). The potential was inverted for 30 s and the lipid analogue was dialyzed for 30 min against RNAse-free water (Sigma). The concentration of peptidyl-RNA adduct was determined by spectrophotometry (ε260 nm = 180,000 M -1 cm -1 ; 232 pmol; yield: 18%) and analyzed by LC-MS.

Synthesis of neryl-PP-disaccharide-peptide-RNA (8b and 8c, Scheme S5). For neryl-containing analogues, the Huisgen-Sharpless cycloaddition reaction was performed in a volume of 10 µl containing lipid II analogues (1 mM), the azido-RNA helix (0.5 mM), tris (3hydroxypropyltriazolylmethyl)amine (THPTA) (3.5 mM), copper sulfate (0.5 mM), sodium ascorbate (10 mM), sodium phosphate (100 mM; pH 8.0). The lipid II analogues and the azido-RNA helix were lyophilized together prior to the addition of the remaining components. Incubation was performed in a PCR thermocycler at 37°C for 2 h. The peptidyl-RNAs were purified by anion exchange chromatography (DNAPAc PA100; Dionex) with a linear gradient (25 mM to 2.5 M) of ammonium acetate (pH 8.0) containing 0.5% acetonitrile (v/v), which was applied between 8.5 and 33.5 min (Figure S2 c andd). The peptidyl-RNA adducts were lyophilized twice and dissolved in RNAse-free water (Sigma). The purity of peptidyl-RNA adducts was assessed by denaturing (7 M urea) polyacrylamide gel electrophoresis (12 %), by anion exchange chromatography, and by LC-MS. The concentration of peptidyl-RNA adducts was determined by spectrophotometry (ε 260 nm = 180,000 M -1 cm -1 ). 8b) or D-Lac (8c). Anionic rpHPLC was performed on an analytical DNAPac PA-100 column (Thermofisher Scientific, 4.6 x 250 mm) at a flow rate of 1 mL min -1 with a linear gradient (0 to 100% B) applied between 8 and 33 min. Buffer A, 25 mM ammonium acetate (pH 8.0) containing 0.5% ACN (v/v). Buffer B, 2.5 M ammonium acetate (pH 8.0) containing 0.5% ACN (v/v). Absorbance was monitored at 260 nm.

Enzymatic assays

Aminoacylation of tRNA Gly by the GlyRS amino-acyl-tRNA synthetase. The acceptor capacity of tRNA Gly obtained by in vitro transcription was evaluated in a volume of 15 µl containing RNA (ca. 3 µM, as determined by spectrophotometry, ε260 nm = 708,000 M -1 cm -1 ), GlyRS (0 to 800 nM), [ 14 C]Gly (3.74 GBq/mmol, 20 µM), ATP (2.5 mM), Tris-HCl (25 mM; pH 7.5), MgCl2 (7.5 mM), KCl (12.5 mM), DTT (2 mM), and BSA (0.1 mg mL -1 ). The reaction mixture was incubated for 8 min at 30°C and the acylated [ 14 C]Gly-tRNA Gly was precipitated by the addition of 1 mL of cold trichloroacetic acid (7%) containing 0.5% casaminoacid (w/v). The precipitate was filtrated on a GF/C Whattmann filter previously washed three times with 1 mL of the TCA-casamino acids solution. The filters were washed three times with 1 mL of the TCA-casamino acids solution and dried at room temperature for 30 min. [ 14 C]Gly-tRNA Gly was determined with a Beckmann Coulter LS6500 liquid scintillation counter (Figure S3).

Figure S3

. Enzymatic determination of tRNA Gly . The reaction mixtures contained 45 pmoles of tRNA Gly based on spectrophotometry (ε260 nm = 708,000 M -1 cm -1 ). The radioactive assay indicates that 32 pmoles (71%) of tRNA Gly , as determined by spectrophotometry, were acylated by GlyRS.

FmhB-GlyRS coupled assay. The FmhB transferase catalyzes the transfer of a glycyl residue from Gly-tRNA Gly to the lipid II peptidoglycan precursor (Figure S4). In this study, Gly-tRNA Gly was generated in the reaction mixture by a high concentration of GlyRS that maintains full acylation of tRNA Gly during the entire time course of the reaction (Figure S3, above). [1] The reaction mixture (25 µL) contained GlyRS (0.8 µM), heptaprenyl-or neryl-containing lipid II analogues (30 µM), [ 14 C]Gly (50 µM; 3.74 GBq/mmol), tRNA Gly (0.4 µM), Tris-HCl (50 mM; pH 7.5), ATP (7.5 mM), MgCl2 (12.5 mM) and DTT (2 mM). The reaction was initiated by the addition of FmhB (0.05 µM and 0.17 µM for heptaprenyl-and neryl-containing lipid II, respectively). The heptaprenyl-containing lipid II analogues were extemporaneously lyophilized prior to dissolution in a 10% Triton TM X-100 solution. The final concentration of Triton TM X-100 in the assay was 1%. The inhibitors were used at concentrations ranging from 0 to 2.5 µM. The reactions were allowed to proceed at 30°C and aliquots (2 µl) were withdrawn at 1, 10, 20, 30, 40, 50, 60, 90, and 120 min. The reactions were stopped at 80°C for 2 min, cooled in ice for 1 min, and centrifuged at 12,000 x g for 5 min. The supernatants were spotted on chromatography paper (Whatman 4MM, Elancourt, France) or on pre-coated cellulose TLC-sheet (ALUGRAM CEL 300; Macherey-Nagel) for heptaprenyl-and neryl-containing lipid II, respectively. Anionic rpHPLC on semi-preparative DNAPac PA-100 (Thermofisher Scientific, 10 x 250 mm) at 3.5 mL min -1 with a 30 min gradient from 25 mM to 2.5 M ammonium acetate pH 8.0 containing 0.5% ACN (absorbance measured at 260 nm).

[M-2H] 2- [M-H] - +Na +2Na +3Na +Na [M-2H] 2- [M-H] -
[M-2H] 2- [M-H] - [M-2H] 2- [M-H] - +Na +2Na +3Na
[M-H] - +Na [M-H] - [M-2H] 2- [M-2H] 2-

UDP-MurNAc

Azido-RNA

Anionic rpHPLC on analytical DNAPac PA-100 (Thermofisher Scientific, 4.6 x 250 mm) at 1 mL min -1 with a 25 min gradient applied between 8 and 33 min from 25 mM to 2.5 M ammonium acetate pH 8.0 containing 0.5% ACN (absorbance measured at 260 nm).

UDP-MurNAc-pentapeptide (1a)

Anionic rpHPLC on analytical DNAPac PA-100 (Thermofisher Scientific, 4.6 x mm) at 1 mL min -1 with a 22 min gradient from 25 mM to 375 mM ammonium acetate pH 8.0 containing 0.5% ACN between 1 and 23 min (absorbance measured at 260 nm).

UDP-MurNAc-pentadepsipeptide (1b)

Anionic rpHPLC on analytical DNAPac PA-100 (Thermofisher Scientific, 4.6 x mm) at 1 mL min -1 with a 22 min gradient from 25 mM to 375 mM ammonium acetate pH 8.0 containing 0.5% ACN between 1 and 23 min (absorbance measured at 260 nm).

Figure 1 .

 1 Figure 1. a) Assembly of the pentaglycine side-chain of peptidoglycan precursors by amino-acyl

Figure 2 .

 2 Figure 2. Semi-synthesis of lipid II analogues. MraY, Phospho-N-acetylmuramoyl-pentapeptide-

Figure 3 .

 3 Figure 3. Catalytic efficacy of FmhB for the transfer of Gly from Gly-tRNA Gly to lipid II analogues ending

CNFigure 4 .

 4 Figure 4. Synthesis of the n-prenyl-carbohydrate-peptidyl-RNA conjugates. X = NH (D-Ala) or X = O (D-

Figure 5 .

 5 Figure 5. Inhibition of FmhB by lipid-carbohydrate-peptidyl-RNA conjugates. (a) Structure of the inhibitors. X = NH (D-Ala) or X = O (D-Lac). (b and c) Separation of [ 14 C]Gly from [ 14 C]Gly-lipid II

  Scheme S2. (i) 1. L-Ala, MurC; 2. D-Glu, MurD; 3. meso-cystine, MurE; (ii) D-Ala-D-Ala or D-Ala-D-Lac, MurF; (iii) DTT, Tris-HCl buffer, pH 8.0, 2 h, RT; (iv) MSH, DMF, 2 h, RT; (v) 3-butyne-1-thiol, DMF, 2 h, 37°C.

Figure S1 .

 S1 Scheme S3. Mechanism for one pot conversion of meso-cystine to dehydroalanine

  Scheme S4. One pot enzymatic synthesis of lipid II analogues. heptaprenyl-phosphate, n = 6; nerylphosphate n = 1.

  Scheme S5. Coupling of lipid II analogues to RNA by click chemistry. Phosphate buffer, 2 h, 37°C; 2'-N3-RNA; CuSO4, THPTA, sodium ascorbate, 2 h. Heptaprenyl-phosphate, n = 6; neryl-phosphate, n = 1.

Figure S2 .

 S2 Figure S2. (a) Upper panels: purification of the heptaprenyl-PP-disaccharide-peptidyl-RNA conjugate (8a) by denaturing polyacrylamide gel electrophoresis. Lane 1, reaction; lane 2, purified azido-RNA (160 pmoles). The red box indicates the portion of the gel containing the band of interest prior (left panel) and after (right panel) excision. Lower panels: Analyses of rpHPLC purifications of the cycloaddition of azido-RNA with alkyne-containing neryl-PP-disaccharide-peptide ending in D-Ala or D-Lac. lane 1, purified azido-RNA (250 pmoles), lanes 2 to 4, rpHPLC fractions from panel b and c. (b) rpHPLC analysis of purified azido-RNA by anionic rpHPLC (RT = 20.6 min). (c and d) Purification of neryl-PP-disaccharide-peptidyl-RNA conjugates ending in D-Ala (8b) or D-Lac (8c). Anionic rpHPLC was performed on an analytical DNAPac PA-100 column (Thermofisher Scientific, 4.6 x 250 mm) at a flow rate of 1 mL min -1 with a linear gradient (0 to 100% B) applied between 8 and 33 min. Buffer A, 25 mM ammonium acetate (pH 8.0) containing 0.5% ACN (v/v). Buffer B, 2.5 M ammonium acetate (pH 8.0) containing 0.5% ACN (v/v). Absorbance was monitored at 260 nm.

Figure S4 .

 S4 Figure S4. Reactions catalyzed by GlyRS and FmhB. The glycyl-tRNA synthetase (GlyRS) acylates tRNA Gly (depicted as a clover leaf). FmhB transfers Gly from Gly-tRNA Gly to the lipid II analogues. Heptaprenylphosphate, n = 6; neryl-phosphate, n = 1.

Figure

  Figure S5. (a) and (b) Kinetics of transfer of [ 14 C]Gly from Gly-tRNA Gly to heptaprenyl-and nerylcontaining lipid II analogues, respectively. The reactions products were analyzed by descending paper chromatography and ascending thin-layer chromatography, respectively.

Figure S6 .

 S6 Figure S6. Inhibition of FmhB by D-Ala-and heptaprenyl-containing disaccharide-peptidyl-RNA conjugate (8a). The IC50 value (5± 3 nM) was deduced by fitting the Morrison equation to the data of three independent experiment.

  rpHPLC on analytical 100-5 C18 Nucleosil column (4.6 x 250 mm; Macherey-Nagel), 10 mM ammonium formate pH 5.0; 30 min linear gradient from 0 to 50% ACN applied between 6.5 and 36.5 min (absorbance measured at 214 nm).D-Lac-and neryl-containing lipid II (3b)rpHPLC 100-5 C18 Nucleosil (4.6 x 250 mm; Macherey-Nagel), 10mM ammonium formate pH 5.0; 30 min linear gradient from 0 to 50% ACN applied between 6.5 and 36.5 min (absorbance measured at 214 nm).

  rpHPLC on analytical 100-5 C18 Nucleosil column(4.6 x 250 mm; Macherey-Nagel), 10 mM ammonium formate pH 5.0; 30 min linear gradient from 0 to 50% ACN applied between 10 and min (absorbance measured at 214 nm).UDP-MurNAc-L-Ala-D-iGlu-cystine-D-Ala-D-Lac (4b)rpHPLC on analytical 100-5 C18 Nucleosil column (4.6 x 250 mm; Macherey-Nagel), 10 mM ammonium formate pH 5.0; 30 min linear gradient from 0 to 50% ACN applied between 10 and min (rpHPLC analytical 100-5 C18 Nucleosil column (4.6 x 250 mm; Macherey-Nagel), 10 mM ammonium formate pH 5.0; 30 min linear gradient from 0 to 15% ACN applied between and 40 min (absorbance measured at 214 nm).rpHPLC on analytical 100-5 C18 Nucleosil column (4.6 x 250 mm; Macherey-Nagel), 10 mM ammonium formate pH 5.0; 30 min linear gradient from 0 to 15% ACN applied between 10 and 40 min (absorbance measured at 214 nm). rpHPLC analytical 100-5 C18 Nucleosil column (4.6 x 250 mm; Macherey-Nagel), 10 mM ammonium formate pH 5.0; 30 min linear gradient from 0 to 50% ACN applied between 6.5 and 36.5 min (absorbance measured at 214 nm). -and neryl-containing disaccharide-peptidyl-RNA conjugate (8b) Anionic rpHPLC on analytical DNAPac PA-100 column (Thermofisher Scientific, 4.6 x 250 mm) at 1 mL min -1 with a 25 min gradient applied between 8 and 33 min from 25 mM to 2.5 M ammonium acetate pH 8.0 containing 0.5% ACN (absorbance measured at 260 nm).D-Lac-and neryl-containing disaccharide-peptidyl-RNA conjugate (8c)Anionic rpHPLC on analytical DNAPac PA-100 column (Thermofisher Scientific, 4.6 x 250 mm) at 1 mL min -1 with a 25 min gradient applied between 8 and 33 min from 25 mM to 2.5 M ammonium acetate pH 8.0 containing 0.5% ACN (absorbance measured at 260 nm).

  ).

			UDP-MurNAc
					NH						NH
					O						O
					HN						HN
					CO 2 H					CO 2 H
	H 2 N	S CO 2 H	S	O	NH O	H 2 N	TCEP Br O	O Br	NH 2	O	NH O
				HN					HN
				O	X CO 2 H					O	X CO 2 H	G 7 A 6 G 1 C 2 G 3 G 5 G 4	U 73 C 72 G 71 C 70 C 69 U 68 U 67 C 66	NH
												C 75 C 74	HN	O
												-	O	P O	O	O	A	CO 2 H
												O	HO N N	O	NH
												N	S	O
												HN
												O
												X
												CO 2 H

  Cys with O-mesitylenesulfonylhydroxylamine (MSH) (20 mM) in DMF-phosphate buffer (50 mM; pH 8.0) (4/6, v/v) for 2 hours at RT. Yield: 28% for 5a (v) Addition of 3-butyne-1-thiol (50 mM) to the resulting dehydroalanyl residue (0.5 mM) in DMF-phosphate buffer (100 mM; pH 8.0) (1/19, v/v) for 2.5 h at 37°C. After addition of 1 volume of buffer A (50 mM ammonium acetate, pH 5.0), the desired isomer (depicted in Scheme 2) was purified by rpHPLC (EC 250/4.6 Nucleosil 100-5 C18;

D-Ala-D-Lac to UDP-MurNActripeptide by synthetase MurF. (iii) Reduction of the cystinyl residue of the resulting UDP-MurNAcpentapeptide and -depsipentapeptide analogues by DTT (20 mM) in Tris-HCl (50 mM; pH 8.0) at RT for 2 h. Yields: 90% for both D-Ala-and D-Lac-ending anlogues. (iv) Oxidation-elimination of the sulfhydryl group of L-

D-Ala-and heptaprenyl-containing Lipid II (2a) D-Lac-and heptaprenyl-containing Lipid II (2b)

	p	g			,	,		,
	T: -p ESI Full ms [440,00-2000,00]
																	1601,84
	Relative Abundance
						800,38
																	1623,46
		509,32	555,16	635,53		766,19	811,36 900,04	947,61	1055,78 1156,97	1245,19	1310,52	1398,40	1566,32	1639,90	1784,86	1856,98	1978,92
		500	600	700	800	900	1000	1100		1200		1300	1400	1500	1600	1700	1800	1900	2000
													m/z
		_			,	,			,
	T: -p ESI Full ms [500,00-2000,00]
																	1602,87
																	1625,73
						801,00
																	1647,87
	Relative Abundance
																	1669,73
																	1726,60
						812,33
																	1744,27
		596,93	764,93 756,00	876,60	964,27	1112,60 1080,87	1187,67	1354,80 1382,67 1307,47	1532,53	1836,00	1928,87
			600	700		800	900		1000		1100	1200	1300	1400	1500	1600	1700	1800	1900	2000
													m/z

D-Ala-and neryl-containing lipid II (3a) D-Lac-and neryl-containing lipid II (3b)

	T: -p ESI Full ms [345,00-2000,00]
													1261,42
	Relative Abundance
				630,27
														1283,44
	412,73									1058,44
	474,60	617,70	641,31		757,41 826,59	947,41	1003,82	1136,88	1238,87	1299,14	1423,08		1570,18	1716,34	1861,04	1931,82
	400	500	600	700	800	900	1000	1100		1200	1300	1400	1500	1600	1700	1800	1900	2000
												m/z
	T: -p ESI Full ms [1000,00-1500,00]
													1262,33
	Relative Abundance
													1284,33
														1300,20
														1306,20
															1344,33
	1028,20	1059,20	1082,07	1125,20	1149,27	1172,47	1212,13	1251,60				1366,07	1391,87	1443,93	1482,93
		1050			1100			1150		1200		1250	1300		1350		1400	1450	1500
												m/z

Ala-and heptaprenyl-containing disaccharide-peptidyl-RNA conjugate (8a) D-Ala-and neryl-containing disaccharide-peptidyl-RNA conjugate (8b)

	Azido-RNA D-p D-Lac		,	,		,
	T: -p ESI Full ms [800,00-1550,00] T: -p ESI Full ms [500,00-2000,00]
		701,20											1289,47
			Z=11								Z=5 Z=5	[M-H] -	1543,80		Z=4 Z=4
				771,53 Z=10													1929,93
					Z=6 Z=6
	Relative Abundance Relative Abundance	Z=7 Z=7		857,27 Z=9	Z=8 964,40		Z=7	Z=6		1311,47 +Na 1467,47		1766,00
		664,33			909,00	1043,47 981,73	1102,33	1239,00 1181,73		1400,67	1593,60	1721,60
		598,20														1627,60	1816,00
															+2Na
															1333,33
		846,67 886,60 836,67 853,07	942,73	997,13	1040,93	1091,67	1135,73	1198,60	1260,33	1355,40 +3Na	1405,73	1431,00	1490,73 1525,07
		850 600	900 700	950 800	1000 900		1050 1000		1100 1100	1150 1200		1200 1300	1250 1400	1300 1500	1350 1600	1400 1700	1450 1800	1500 1900	1550 2000
											m/z m/z
	T: -p ESI Full ms [800,00-1550,00] T: -p ESI Full ms [800,00-1550,00]				1053,53 Z=7	Z=18
									1334,87
														Z
				1264,53 Z=19							1229,13	1413,27
													1257,27			1501,40 1475,13
		Z=20
	Relative Abundance Relative Abundance	1202,47 1218,53 839,40 913,67 1236,00 1245,40 819,27 933,87 941,73 968,73 1273,60 1294,13 1298,40 984,20 1017,07 1306,93 1327,40 1084,27 1077,00 1119,40 1135,40 1354,60 959,33 921,73 Z=8	1393,40 1386,27	1444,20 1433,73 1287,67 1360,60 1462,53 1473,40 1328,87 1276,27 1377,87 1385,40	1436,53 1491,60	1529,40 1518,73 1535,93 1507,93
											1192,00
		864,80															1531,73
		898,47														1407,33
		1200		1250			1300			1350			1400		1450	1500
		850	900	950	1000		1050		1100	1150	m/z	1200	1250	1300	1350	1400	1450	1500	1550
											m/z

-and neryl-containing disaccharide-peptidyl-RNA conjugate (8c)

	8-
	p			,	,	,
	T: -p ESI Full ms [800,00-1550,00]
																1475,73
										Z=6
										1229,33
	Relative Abundance					1053,87 Z=7
																1511,80
			921,87			1077,53	1111,40			1259,53
	836,80	857,87	983,00 953,60 936,27					1209,27	1270,00	1311,13
	820,93		913,33		1027,00		1127,47	1182,87				1350,87 1385,93	1425,73	1460,87	1531,33
	850	900	950	1000	1050	1100	1150	1200	1250	1300	1350	1400	1450	1500	1550
										m/z

rpHPLC analysis of synthesized compounds tRNA Gly

Acknowledgements

This work was supported by the project SyntRNA from ANR program to MEQ and MA (ANR-17-CE07-0041-01). NS was funded by a DGA-MRIS scholarship.

Supplementary information

Synthesis of lipid-carbohydrate-peptidyl-RNA conjugates to explore the limits imposed by the substrate specificity of cell wall enzymes on the acquisition of drug resistance


G Durif

L Modolo

J E Mold

S Lambert-Lacroix

F Picard

Probabilistic Count Matrix Factorization for Single Cell Expression Data Analysis

The combination of massive parallel sequencing with high-throughput cell biology technologies has given rise to single-cell Genomics. Similar to the paradigm shift of the 90s characterized by the first molecular profiles of tissues, it is now possible to characterize molecular heterogeneities at the cellular level [START_REF] Saliba | Single-cell RNAseq: Advances and future challenges[END_REF]. The statistical characterization of heterogeneities in single-cell expression data thus requires an appropriate model, since the transcripts abundance is quantified for each cell using read counts. Hence, standard methods based on Gaussian assumptions are likely to fail to catch the biological variability of lowly expressed genes, and Poisson or Negative Binomial distributions constitute an appropriate framework [START_REF] Chen | Detection of high variability in gene expression from single-cell RNA-seq profiling[END_REF]. Moreover, dropouts, either technical (due to sampling difficulties) or biological (no expression or stochastic transcriptional activity), constitute another major source of variability in scRNA-seq (single-cell RNA-seq) data, which has motivated the development of the so-called Zero-Inflated models [START_REF] Kharchenko | Bayesian approach to single-cell differential expression analysis[END_REF]. A standard and popular way of quantifying and visualizing the variability within a dataset is dimension reduction, principal component analysis (PCA) being the most widely used technique in practice. Model-based PCA [START_REF] Collins | A generalization of principal components analysis to the exponential family[END_REF] offers the unique advantage to be adapted to the data distribution and to be based on an appropriate metric, the Bregman divergence. It consists in specifying the distribution of the data through a statistical model. A probabilistic zero-inflated version of the Gaussian PCA was proposed by [START_REF] Pierson | ZIFA: Dimensionality reduction for zero-inflated singlecell gene expression analysis[END_REF] in the context of single cell data analysis (the ZIFA method). However, scRNA-seq data may be better analyzed by methods dedicated to count data such as the Nonnegative Matrix Factorization (Lee & Seung, 1999, NMF) or the Gamma-Poisson factor model [START_REF] Cemgil | Bayesian inference for nonnegative matrix factorisation models[END_REF]. However, none of the currently available dimension reduction methods fully model single-cell expression data, characterized by overdispered zero inflated counts [START_REF] Zappia | Splatter: Simulation of single-cell RNA sequencing data[END_REF]. Our method is based on a probabilistic count matrix factorization (pCMF). We propose a dimension reduction method that is dedicated to over-dispersed counts with dropouts, in high dimension. Our factor model takes advantage of the Poisson Gamma representation to model counts from scRNA-seq data [START_REF] Zappia | Splatter: Simulation of single-cell RNA sequencing data[END_REF]. In particular, we use Gamma priors on the distribution of principal components. We model dropouts with a Zero-Inflated Poisson distribution, and we introduce sparsity in the model thanks to a spike-and-slab approach [START_REF] Malsiner-Walli | Comparing spike and slab priors for Bayesian variable selection[END_REF] that is based on a two component sparsity-inducing prior on loadings [START_REF] Titsias | Spike and slab variational inference for multi-task and multiple kernel learning[END_REF]. The model is inferred using a variational EM algorithm that scales favorably to data dimension, as compared with Markov Chain Monte Carlo (MCMC) methods [START_REF] Blei | Variational inference: A review for statisticians[END_REF]. Then we propose a new criterion to assess the quality of fit of the model to the data, as a percentage of explained deviance, because the standard variance reduction that is used in PCA needs to be adapted to the new framework dedicated to counts. We show that pCMF better catches the variability of simulated data and experimental scRNA-seq datasets. Finally, pCMF is available in the form of a R package available at https://gitlab.inria.fr/gdurif/pCMF.


Alison E Turnbull
email: turnbull@jhmi.edu

Sarina K Sahetya

E Lee

Daugherty Biddison

Christiane S Hartog

Gordon D Rubenfeld

Dominique D Benoit

Bertrand Guidet

Rik T Gerritsen

Mark R Tonelli

Randall Curtis

J Randall Curtis

Competing and conflicting interests in the care of critically ill patients

Keywords: Critical care, Patient-centered care, Research design, Clinical studies as topic, Conflict of interest

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

Despite often meeting for the first time at admission, most hospitalized patients trust clinicians with their lives. In the intensive care unit (ICU), this trust is born of necessity-after all, most ICU patients are not stable enough to seek care elsewhere. However, it is naïve for clinicians to assume patient trust is invulnerable or entirely rational, especially as public trust in scientific, political, religious, and media institutions falters in some parts of the world [START_REF] Gauchat | Politicization of science in the public sphere: a study of public trust in the United States, 1974 to 2010[END_REF][2][START_REF] Funk | Mixed messages about public trust in science[END_REF].

Trust is the belief that another person or entity will act in our best interest [START_REF] Cook | Trust in Society[END_REF][START_REF] Hardin | Trust and Trustworthiness[END_REF]. Public trust in the medical profession stems in part from policies instructing healthcare providers to prioritize patient interests above other interests. For example, the American Medical Association (AMA) states in its Code of Medical Ethics [START_REF]AMA code of medical ethics[END_REF] that "physicians' ethical responsibility [is] to place patients' welfare above the physician's own self-interest" (Opinion 1.1.1). As a result, unless they belong to a population that has historically been harmed by medical professionals [START_REF] Gamble | Under the shadow of Tuskegee: african Americans and health care[END_REF][START_REF] Jones | The health care experiments at many farms: the Navajo, Tuberculosis, and the Limits of modern medicine, 1952-1962[END_REF][START_REF] Stern | Sterilized in the name of public health: race, immigration, and reproductive control in modern California[END_REF], patients and their families rarely question clinician motives. But while patient welfare, or well-being, is a clinician's primary interest, competing interests are ubiquitous [START_REF] Stell | Two cheers for physicians' conflicts of interest[END_REF]. Managing the competing interests of other patients, clinicians, hospital administrators, trainees, and one's own implicit biases has become so routine that we often forget this balancing act is ever-present and yet generally invisible to patients.

Interests that influence physician decision-making and compete with patient well-being generate conflicts of interest [START_REF] Schünemann | An official American Thoracic Society Policy statement: managing conflict of interest in professional societies[END_REF][START_REF] Mccoy | Why there are no "potential" conflicts of interest[END_REF], defined as "circumstances that create a risk that professional judgments or actions regarding a primary interest will be unduly influenced by a secondary interest" [START_REF]Committee on Conflict of Interest in Medical Research, Education, and Practice[END_REF] (Fig. 1). What constitutes "undue" influence, and therefore a conflict of interest, is subjective and context specific. Similarly, well-being encompasses how a patient thinks and feels about his or her life and is patient-specific [START_REF] Diener | The evolving concept of subjective well-being: The multifaceted nature of happiness[END_REF]. Competing interests alone do not necessarily constitute an ethical problem or result in harm to patients, but they do produce conditions that increase the risk of harm to patients [START_REF] Mccoy | Why there are no "potential" conflicts of interest[END_REF]. The seriousness of the harm that can result when a conflict of interest impacts a decision about the care of a critically ill patient creates a special obligation for intensivists to develop both a high degree of situational awareness and self-awareness and the courage to name and discuss the competing interests they continually work to manage.

Ideally, conflicts of interest should be prevented through policies and practices that align clinician and patient interests. This idea underlies the recent formation of accountable care organizations (ACOs) in the USA as part of the Affordable Care Act [START_REF] Mcclellan | A national strategy to put accountable care into practice[END_REF]. Physicians participating in ACOs make more money if they keep patients healthy rather than if they provide more treatments. Similarly, the organization of some novel healthcare financing systems in Europe intends to strengthen patient-specific decision-making [START_REF] Pimperl | Evaluating the impact of an accountable care organization on population health: the quasi-experimental design of the German Gesundes Kinzigtal[END_REF]. When secondary competing interests cannot be eliminated, the risk that they will influence a physician's judgment should be minimized via procedures that allow decisions to be periodically reviewed for evidence of undue influence. Conflicts of interest are ubiquitous throughout medicine, but the potential consequences for critically ill patients who generally lack the ability to advocate for themselves or seek care elsewhere make it important to explicitly address this issue in the ICU setting.

Here, we explore factors that increase the risk, or the perception, that an ICU physician will reason, recommend, or act in a way that might be construed as not in their patient's best interest [START_REF] Tonelli | Conflict of interest in clinical practice[END_REF]. Our goal is not to dishonor or cast doubt on prior decisions. Rather, we hope that naming the many parties with a stake in these decisions and the pervasive competing interests will illustrate both the ethical challenges intensivists are routinely expected to navigate and the vast faith patients and families place in the integrity of critical care professionals. Understanding, exploring, and explicitly addressing competing interests provide an opportunity to minimize the impact of these interests on patient care, thereby potentially preventing avoidable harm.

Interests of other ICU patients and patients being considered for ICU admission

Resources such as operating rooms, equipment, and physicians' or other clinicians' time and attention are finite. So while patient well-being is an intensivist's first priority, not every patient can be first. When multiple ICU patients stand to benefit from a limited resource, intensivists decide which patients to treat first [START_REF] Evans | Influence of ward round order on critically ill patient outcomes[END_REF] (Table 1). These decisions constitute a form of triage in which priority is generally given to patients who are most likely to benefit or are at the greatest risk of death. Managing the competing interests of admitted patients is generally not recognized as an ethical problem unless ethically irrelevant factors such as patient race, gender, religion, political preferences, citizenship status, ability to pay for care, celebrity status, personal relationships with hospital personnel, or sexual orientation influence decisions [START_REF] Stepanikova | Racial-ethnic biases, time pressure, and medical decisions[END_REF][START_REF] Fitzgerald | Implicit bias in healthcare professionals: a systematic review[END_REF]. However, because ICU patients are unaware of other patients' needs, how patients are prioritized is not transparent, and in the context of promotional campaigns that stress the philosophy of patient-centered care, waiting for care or attention is sometimes surprising or confusing for patients and their families.

The rationales supporting decisions about which patients to admit to an ICU differ across countries [START_REF] Evans | Critical care rationing: international comparisons[END_REF], are generally not communicated to patients [START_REF] Mielke | Priority setting in a hospital critical care unit: qualitative case study[END_REF][START_REF] Cooper | Communication of bed allocation decisions in a critical care unit and accountability for reasonableness[END_REF], and can impact the care of existing ICU patients. This is most

Unethical acƟons

RecommendaƟons or acƟons which harm paƟents and benefit a secondary interest.

Conflicts of Interest

Circumstances where there is risk clinician acƟons will be unduly influenced by the secondary interest.

CompeƟng Interests

Secondary interests that compete with a paƟent's best interest.

Ubiquitous and unavoidable

Describes the concern of someone other than the paƟent

Interests of family members

Patients' family members are usually assumed to advocate for their loved one's best interests. But a patient's death can have major financial ramifications for families that motivate decisions. For example, when a recipient of disability benefits dies in the US, the Social Security Administration generally reduces or stops issuing payments. If the patient's family is financially dependent on these disability payments, death becomes a harbinger of destitution. Conversely, family members of patients with substantial wealth or a generous life insurance policy stand to benefit financially from a patient's death. When patients decide to forgo or end treatment, some family members are unable to accept the decision. In this situation, patients may acquiesce to another round of chemotherapy or a few more weeks of mechanical ventilation to give their loved ones time to accept their prognosis. But other patients are adamantly opposed to additional treatment. Even when a patient has been unwavering and has legally documented their preferences, a power differential remains between terminally ill patients and their families. This imbalance stems from the fact that patients with critical illness often lose the ability to communicate or advocate for themselves. Clinicians know such patients are unlikely to regain sufficient strength to create bad publicity for the hospital or file a lawsuit for assault, battery, or inadequate informed consent [START_REF] Pope | Legal briefing: new penalties for disregarding advance directives and do-not-resuscitate orders[END_REF]. In contrast, distraught or aggrieved family members may register complaints with the hospital's office of patient relations, post scathing reviews online, file a wrongful death lawsuit, or even threaten physical violence. Even intensivists who believe a patient's decision to decline interventions is in that patient's best interest sometimes acquiesce to insistent family members in the US, although this is illegal in some European countries [START_REF] Abadir | When doctors and daughters disagree: twenty-two days and two blinks of an eye[END_REF]. Recognizing when families constitute a competing interest to patient well-being offers the opportunity to strive for more informed and ethical decisionmaking in these taxing situations.

Interests of medical trainees

For teaching hospitals, medical education remains a core component of the tripartite mission. However, most patients do not understand the gradations of training within members of the medical team, and they are rarely asked to consent to involving physician trainees in their care. Teaching hospitals offer access to state-of-the art technology and renowned clinicians. Many teaching hospitals in the US also offer non-teaching services for patients who do not want to be treated by trainees [START_REF] Reilly | Don't learn on me-are teaching Hospitals Patient-Centered?[END_REF]. Outside these non-teaching wards, faculty in teaching hospitals attempt to ensure patients receive appropriate care from the most capable physician while also ensuring their trainees receive appropriate experience in directly caring for patients. If this balance is not appropriately achieved, unnecessary interventions may be performed primarily for "medical education" rather than the benefit

Table 1 How the interests of other patients, family members, and trainees compete or conflict with patient interests CVVHD Continuous veno-venous hemodialysis, ICU intensive care unit

Interested party How a patient's care could be affected

Other ICU patients When multiple patients need a procedure or intervention requiring a particular clinician or piece of equipment, some patients are forced to wait. Example: there are more patients requiring CVVHD than CVVHD units available in the hospital Patients outside the hospital When the ICU is full, patients for whom an additional day of ICU-level care is optimal may be downgraded to make an ICU bed available for a patient seeking ICU admission ICU families Families may gain financially from hastening (inheritance) or delaying (disability benefits) a patient's death. When family members do not support a patient's informed decision to forgo life support, therapies are initiated or continued to assuage family members Medical trainees Critical care fellows, who are not the most experienced clinicians available, must practice certain procedures a predetermined number of times to demonstrate proficiency. Example: Choosing to perform a bronchoscopy for a patient with community-acquired pneumonia when either a bronchoscopy or an endotracheal aspirate is appropriate and fellows need to practice bronchoscopy of the patient, or procedures may result in complications that would not have occurred if performed by a more experienced physician. Explicitly naming these competing interests with trainees should be a routine part of medical education.

Interests of physicians

In both Europe and the US, some intensivists work on a fee-for-service contract under which performing more procedures leads to more financial compensation. When intensivists in the US are salaried, clinical work quotas defined by relative value units (RVUs) are common (Table 2). A payment formula for every test and procedure covered by the US Medicare system has an associated number of RVUs for physician work [START_REF] Laugesen | The resource-based relative value scale and physician reimbursement policy[END_REF]. Many US intensivists must bill for services associated with sufficient RVUs to fulfill their contract, and performing more than the minimum quota may result in a financial bonus. Under this system, performing tests and procedures remains in a physician's financial interest.

In some countries where physicians receive modest government salaries, patients and families have traditionally provided physician bonuses or given expensive gifts [START_REF] Holt | Slovak bribery case sparks wider debate in eastern Europe[END_REF]. While some physicians in the former Soviet Republics view these "irregular payments" as an acceptable way for patients to express gratitude for good care and augment under-funded healthcare systems, others describe the system as medical bribery [START_REF] Kakuk | Healthcare financing and conflict of interests in hungary: the system of irregular payments and its challenges to the integrity of healthcare ethics[END_REF][START_REF] Manea | Medical Bribery and the ethics of trust: the Romanian case[END_REF]. The difference between accepting unregulated payments from patients and illegal payments is also easily muddled. In these environments, it is perhaps unsurprising, then, that writing prescriptions in return for illegal bribes from pharmaceutical companies remains a problem [START_REF] Burashnikova | Attitudes to pharmaceutical promotion techniques among healthcare professionals in the Republic of Tatarstan, Russia[END_REF][START_REF] Jofre | GSK faces accusations of bribing doctors in Poland[END_REF].

An intensivist, like any other clinician, may also consider his or her legal risk when developing a care plan. Most US physicians are involved in a malpractice claim at some point in their career [START_REF] Jena | Malpractice risk according to physician specialty[END_REF]. In response, physicians order tests and procedures, or avoid high-risk patients or procedures, out of fear of malpractice litigation. This practice is referred to as "defensive medicine" [START_REF] Mello | National costs of the medical liability system[END_REF]. The degree to which concern about legal risk impacts physician decisions is challenging to measure, but US hospitalists reported that 28% of their orders were at least partially defensive in a 2008 study [START_REF] Rothberg | The cost of defensive medicine on 3 Hospital Medicine Services[END_REF]. After a lifethreatening medical error has occurred, providers have a strong interest in avoiding being charged with a wrongful death, which is likely to influence decisions about the continued use of life-sustaining (or death-prolonging) therapies.

Intensivists conducting clinical research have a strong interest in enrolling patients in their studies. Enrolling ICU patients is difficult because most patients can not consent, and family members who can provide surrogate consent may be difficult to contact [START_REF] Burns | Research recruitment practices and critically Ill patients[END_REF]. As a result, slow recruitment is the most frequent reason that randomized clinical trials are stopped prematurely in critical care [START_REF] Schandelmaier | Premature discontinuation of randomized trials in critical and emergency care: a retrospective cohort study[END_REF]. Discontinuing a trial because of slow recruitment can have important professional consequences.

Table 2 How physician interests compete with ICU patient interests

Secondary interest

How a patient's care could be affected

Work quotas

Intensivist salaries may require billing patients for a minimum number of relative value units (RVUs) creating an incentive to perform tests and procedures

Legal risk

Diagnostic tests or imaging may be ordered out of concern about malpractice litigation. After an error, fear of a wrongful death suit may drive life-prolonging treatment Study recruitment or outcome Intensivists who are also clinical investigators have an interest in enrolling eligible patients in studies to meet recruitment targets and in study results. Example: If a patient is enrolled in a study with a primary outcome of extubation failure, intensivists may attempt to delay re-intubation with additional breathing treatments or non-invasive ventilation, while a similar patient not enrolled in the study is reintubated earlier

Conflict avoidance

Patients and their family experience high emotional distress, often expressed as sadness or anger when a poor prognosis is disclosed. Disclosure of prognosis is delayed to avoid witnessing and responding to this reaction Maintaining team relations Treatment decisions prioritize work flow rather than patient welfare. Example: A Foley catheter remains in place for a patient to increase ease of nursing care

Avoiding bureaucracy

Intensivists may be less likely to prescribe therapies requiring an approval process. Example: An intensivists waits until 22:01 to place an order for an antibiotic to avoid the additional forms and phone calls required for antibiotic approval when antibiotic stewards are on-duty Curiosity Desire to understand the pathophysiology of disease may influence diagnostic testing or imaging decisions even if results aren't expected to impact patient care Fame Unusual treatments are more likely to garner positive media attention than the standard of care

Religious belief

Intensivists who adhere to a religious tradition that condemns withdrawing life support may avoid discussing this option with patients or family members who are requesting evaluation of the goals of care Financial gain Intensivists may preferentially prescribe or utilize products from companies they have invested in or that provide financial support through grants or honoraria

Sponsors may be less likely to fund future trials proposed by the investigator, the ICU may be viewed as a lessdesirable partner in future multi-site trials, and an academic clinical investigator's prospects for professional advancement dim. Therefore, if a patient must receive a particular treatment or intervention to be eligible for a trial, the prospect of being able to recruit the patient can create an incentive for treatment. While Institutional Review Boards (IRBs) provide oversite of how trials are advertised and consent is obtained, they generally do not have the ability to monitor whether triage or treatment decisions are being made to increase the number of potentially eligible patients. The research practices and elements of informed consent required by IRBs also vary considerably across institutions [START_REF] Silverman | Variability among institutional review boards' decisions within the context of a multicenter trial[END_REF][START_REF] Khan | Variability of the institutional review board process within a national research network[END_REF]. Additionally, when trials are not blinded, desire to demonstrate the intervention's effectiveness may impact treatment decisions. For example, prior research has suggested that there is a higher threshold for initiating Do Not Resuscitate (DNR) orders for ICU patients enrolled in clinical trials [START_REF] Mehter | Do not resuscitate" decisions in acute respiratory distress syndrome: a secondary analysis of clinical trial data[END_REF].

Maintaining interpersonal relationships with patients, patients' family members, colleagues, and administrators is also in a physician's interest. Patients and their families understandably have strong emotional reactions when a poor prognosis is disclosed, and witnessing their distress or anger is uncomfortable. When physicians are busy or unprepared to respond to these emotions, the desire to maintain a cordial relationship with a patient or their family may influence decisions about when to disclose prognosis and inform patients of the option to prioritize comfort rather than longevity [START_REF] Darmon | Formal academic training on ethics may address junior physicians' needs[END_REF]. Maintaining relationships with colleagues in nursing is also a consideration. For example, removing urinary catheters or peripheral intravenous access as soon as possible is generally in the best interest of patients because this decreases the risk of infection. But removing these interventions also creates more work for busy bedside nurses who already struggle with high rates of burnout and moral distress [START_REF] Rushton | Burnout and resilience among nurses practicing in high-intensity settings[END_REF]. Finally, hospitals sometimes require administrative approval before prescribing or performing certain therapies. When the process for requesting approval is burdensome, bureaucratic, or contentious, this creates an incentive for intensivists to seek alternatives rather than advocate for the therapy they believe is in the patient's best interest.

Physician curiosity or desire for recognition can also compete with patient interests. A desire to understand the complex pathophysiology underlying illness draws many physicians into the profession. Understandably, these physicians may desire to perform tests and imaging on patients representing unusual cases, even when the results of testing are not expected to impact treatment decisions and are associated with risks. Of course, some patients and family members share this curiosity and are happy to undergo testing in pursuit of a diagnosis even when it is unlikely to impact treatment. But for critically ill patients who prioritize comfort and time outside the hospital, this testing for the sake of discovery can be burdensome. Unusual cases and treatments can also draw significant media attention. For example, an unorthodox sepsis treatment combining hydrocortisone, vitamin C, and thiamine [START_REF] Marik | Hydrocortisone, vitamin C, and Thiamine for the treatment of severe sepsis and septic shock: a retrospective before-after study[END_REF] has generated substantial media attention [START_REF] Simpson | doctor found a treatment for sepsis. Now he's trying to get the ICU world to listen[END_REF][START_REF]Did An IV Cocktail Of Vitamins And Drugs Save This Lumberjack From Sepsis? In: NPR[END_REF], celebrity for investigators, and funding for a multicenter randomized clinical trial.

Finally, a clinician's religious belief has been described as a competing interest in medicine [START_REF] Smith | Why religious belief should be declared as a competing interest[END_REF]. Currently, most religious institutions condemn physician-assisted death despite its being legal in seven European countries and seven US states [START_REF] Emanuel | Attitudes and practices of euthanasia and physician-assisted suicide in the united states, canada, and europe[END_REF][START_REF] Pope | Legal history of medical aid in dying: physician assisted death in US courts and legislatures[END_REF]. Most ICU patients are too ill to complete the steps required to obtain approval for physician-assisted death, but opposition to the practice can influence a physician's feelings about other practices including palliative sedation and withdrawal of mechanical ventilation. With the exceptions of Sweden, Finland, and Iceland [START_REF] Fiala | Yes we can! Successful examples of disallowing "conscientious objection" in reproductive health care[END_REF], most countries, professional societies [START_REF] Lewis-Newby | An official American Thoracic Society policy statement: managing conscientious objections in intensive care medicine[END_REF], and hospitals have conscientious objection policies permitting clinicians who object to professionally contested interventions on religious grounds to avoid directly participating in those interventions as long as they provide accurate information and a timely referral to a colleague who will perform the intervention [START_REF] Stahl | Physicians, not conscripts-conscientious objection in health care[END_REF].

Interests of hospitals

The interests and concerns of hospital boards and administrators may differ from those of both clinicians and patients. Administrators are responsible for ensuring a hospital remains financially solvent and for advancing the mission of the institution, which may focus exclusively on providing medical care for a local population or balancing patient care, teaching, and research. Depending on the environment, hospital administrators may also feel compelled to consider the interests of the national healthcare system, regulators, political leaders, local employers, insurers, or hospital board members, and this can produce actions that would be in conflict with patients' interests.

The primary interest of any hospital is to continue existing and pursuing its mission. To achieve this, the organization must have sufficient funds to continue operating. Which patients are most advantageous to admit and what treatments are beneficial to offer from a hospital perspective depend on how the hospital is compensated for services (Table 3). In a capitated system where a fixed amount of money is provided per patient, or to care for a population within a given time frame, it is in a hospital's interest to avoid admitting costly patients or providing costly treatments. In contrast, a fee-forservice payment model incentivizes admitting as many patients as possible and performing costly procedures. In countries where hospital revenue depends on patients' ability to pay, or the price charged for care depends on a patient's insurance status, it is in a hospital's interest to admit wealthy or well-insured patients. Administrators often seek to shield providers from these considerations to avoid creating obvious conflicts of interest that could compromise patient care. But observational data and qualitative interviews suggest this shielding may be incomplete. For example, German physicians reported feeling increasing pressure to consider their hospitals' economic interests when making clinical decisions [52]. Similarly, a recent analysis found most patients who received ECMO for Acute Respiratory Distress Syndrome (ARDS)-an unproven but profitable intervention for hospitals [START_REF] Blum | Clinical and billing review of extracorporeal membrane oxygenation[END_REF]-did not first have a trial of prone positioning, which is considered a proven intervention for ARDS but is generally not billable [START_REF] Li | Unproven and expensive before proven and cheap: extracorporeal membrane oxygenation versus prone position in acute respiratory distress syndrome[END_REF].

Hospital administrators must also concern themselves with a hospital's reputation as assessed by quality metrics, rankings, and public opinion. One of the most common quality metrics in the US is the 30-day mortality rate [START_REF] Schwarze | Beyond 30-day mortality: aligning surgical quality with outcomes that patients value[END_REF]. However this metric does not take patient preferences into account, creating an incentive to continue lifeprolonging treatments in patients with poor prognoses who wish to withdraw life support before 30 days postoperatively [START_REF] Walkey | Accounting for patient preferences regarding life-sustaining treatment in evaluations of medical effectiveness and quality[END_REF]. This conflict is even more pronounced in transplant patients. Organ transplant programs are evaluated on the number of patients alive with a functioning transplanted organ at specific time intervals after transplant. One-year survival is publicly reported for all programs in the US, and people who need an organ may choose the transplant center where they wish to be waitlisted. Thus, even when a transplant recipient becomes ICU-dependent and wishes to withdraw life support, US transplant programs have an interest in ensuring the patient remains alive for 1 year.

Finally, hospital reputation and reimbursement may be impacted by measures of patient satisfaction. Such measures are designed to elevate the importance of patient experience, which is laudable and an important outcome in its own right [START_REF] Nash | Why physicians hate "patient satisfaction" but shouldn't[END_REF]. But high satisfaction scores are also associated with greater prescription drug expenditures and higher mortality rates [START_REF] Fenton | The cost of satisfaction: a national study of patient satisfaction, health care utilization, expenditures, and mortality[END_REF], causing some to worry these metrics encourage inappropriate prescribing of drugs with high abuse potential including opioids [START_REF] Zgierska | Patient satisfaction, prescription drug abuse, and potential unintended consequences[END_REF]. Satisfaction scores also create a disincentive for physicians to confront patients about dangerous behaviors such as drug and alcohol abuse or to share bad news about the prognosis, with some physicians reportedly foregoing these conversations as a result [START_REF] Johnston | Patient satisfaction and its discontents[END_REF].

The role of cognitive biases

Cognitive biases are automatic psychologic mechanisms that short-cut deliberate reasoning. While this may be helpful for decision-making under uncertainty, cognitive biases may aggravate competing or conflicting interests. For instance, reciprocity bias is a sense of obligation and can be triggered by presents or financial payments. Wish and confirmation biases lead to selective uptake of information, while commitment and consistency bias restrict our willingness to change our behavior even in the face of new evidence. Implicit bias, any form of cognitive bias about which we have no conscious awareness, may be particularly difficult to address [START_REF] Fitzgerald | Implicit bias in healthcare professionals: a systematic review[END_REF][START_REF] Blumenthal-Barby | Cognitive biases and heuristics in medical decision making: a critical review using a systematic search strategy[END_REF][START_REF] Saposnik | Cognitive biases associated with medical decisions: a systematic review[END_REF]. Some of the more common biases, such as overconfidence and tolerance to risk and uncertainty, may make it more difficult

Table 3 How institutional interests compete with ICU patient interests

Secondary interest

How a patient's care could be affected

Hospital revenue

Hospitals with capitated payment models have an incentive to avoid costly treatments and avoid admitting costly patients Hospitals with fee-for-service payment models have an incentive to perform more costly procedures and admit as many patients as possible In health systems where hospital revenue depends on patients' ability to pay or their insurer, there is a strong incentive to admit patients with health insurance or the ability to directly pay full price Hospital reputation Questions on a publicly available patient satisfaction survey, which can effect hospital reputation and/or reimbursement, often ask about pain management and satisfaction with clinician communication creating incentives for physicians to treat pain aggressively and avoid discussing topics patients and families do not want to hear about

Program reputation

Organ transplant programs in the US are publicly evaluated on volume and on the number of patients alive with a functioning transplanted organ 1 year after transplant. Because people waiting for an organ may choose the transplant center where they wish to be wait-listed, using life-prolonging therapy for at least 1 year after transplant is in the best interest of the transplant program

Ensuring ICU beds are full In some areas, there is pressure to keep ICU beds full to ensure adequate ICU reimbursement and several empty beds might incentivize admitting an elderly patient with multi-morbidity to the ICU when that might not be in the patient's best interest

for clinicians to accurately assess the degree to which a competing interest exists or its potential for harm. Explicit exploration of how these biases interact with or exacerbate the challenge of preventing competing interests from exerting undue influence on patient care can become part of routine clinical care in an environment that is supportive of such exploration. Interactions with the pharmaceutical and device manufacturers in particular may give rise to cognitive biases regarding use of relevant pharmaceuticals and devices [START_REF] Hartog | Systematic analysis of hydroxyethyl starch (HES) reviews: proliferation of low-quality reviews overwhelms the results of well-performed meta-analyses[END_REF][START_REF] Wood | Influence of pharmaceutical marketing on Medicare prescriptions in the District of Columbia[END_REF]. Physicians should actively avoid, and be protected from, exposure to direct and indirect marketing efforts of drug and device manufacturers.

Recommendations

Competing interests in the ICU setting will always exist to varying extents. No clinician can, or should, focus solely on the well-being of one patient without concern for other patients, colleagues, or themselves. But to continue enjoying high levels of public trust, particularly about the use, withholding, and withdrawal of life-support technologies, professional societies should demonstrate commitment to ensuring competing interests do not unduly influence physician judgment or harm patients. The two primary ways to address conflicts of interest are avoidance and disclosure [START_REF] Tonelli | Conflict of interest in clinical practice[END_REF]. A comprehensive approach to conflicts of interest depends on a combination of both avoidance and disclosure, and implementation of this approach must be adapted to the specific circumstances (Fig. 2).

Although routinely mandated in many healthcare settings, the limitations of disclosure are well documented [START_REF] Rodwin | Physicians' conflicts of interest[END_REF]. Asking clinicians to disclose potential conflicts of interest to patients in the ICU setting is often impractical and inappropriate for multiple reasons. First, it places the onus of responsibility on individual clinicians to determine whether they are unduly influenced by a competing interest. This level of objective self-assessment is often not feasible. Social science research has demonstrated that even motivated individuals are unable to remain objective about conflicts of interest because selfserving bias is unintentional [START_REF] Dana | A social science perspective on gifts to physicians from industry[END_REF]. Second, patients and their families struggle to understand the benefits and drawbacks of medical procedures [START_REF] Wegwarth | The barrier to informed choice in cancer screening: statistical illiteracy in physicians and patients[END_REF] and billing under normal circumstances and are even less likely to understand complex professional and financial relationships in the midst of critical illness [START_REF] Netzer | Recognizing, naming, and measuring a family intensive care unit syndrome[END_REF]. Third, most ICU patients are involuntary consumers. Unlike patients seeking elective procedures or an outpatient provider, they cannot choose their provider or decide to transfer to another institution or another physician after learning of a conflict of interest. Finally, disclosure can have adverse and paradoxical unintended consequences. Specifically, disclosure makes some people feel they have moral license to act in their own best interest [START_REF] Cain | The dirt on coming clean: perverse effects of disclosing conflicts of interest[END_REF][START_REF] Loewenstein | The unintended consequences of conflict of interest disclosure[END_REF]. In addition, disclosure of immutable competing interests may serve to erode patient or family member trust in clinicians and thereby make shared medical decision-making more difficult without providing benefit. Determining whether competing interests have undue influence requires explicit exploration of the competing interests and influences as well as data to support this exploration [START_REF] Lo | The future of conflicts of interest: a call for professional standards[END_REF]. Ideally, empiric evidence should inform decisions about when secondary interests are adversely impacting patient care and need to be eliminated or disclosed. In some circumstances, hospitals may be able to monitor themselves, essentially creating a transparent quality improvement program for monitoring and mitigating conflicts of interest. For example, data on ethics committee consultations regarding disagreements on whether to offer cardiopulmonary resuscitation in the event of cardiac arrest at one hospital found that patients who were not offered CPR were more than three times as likely to be critically ill, but there was no association between the committee's recommendation and patient age, race, or functional status at admission [START_REF] Courtwright | Experience with a hospital policy on not offering cardiopulmonary resuscitation when believed more harmful than beneficial[END_REF]. These findings are reassuring for patients, demonstrate the hospital's commitment to ethical practice, reduce the likelihood that biases are permitted to influence decisions, and provide a strong defense against accusations of discrimination. Similarly, Institutional Review Boards and the Scientific Registry of Transplant Recipients monitoring programs could require data on the timing and results of DNR decisions in patients enrolled in trials or receiving organs to safeguard patients from inappropriate pressure.

Exploring and addressing competing and conflicting interests should be an activity supported by the entire ICU team and not be the sole responsibility of individual clinicians. The ethical decision-making climate of an ICU can either support or detract from such exploration. Ethical climate, defined as "individual perceptions of the organization that influences attitudes and behavior and serves as a reference for employee behavior" [START_REF] Olson | Hospital nurses' perceptions of the ethical climate of their work setting. Image[END_REF], should be recognized as an important characteristic of the quality of ICU care that will influence the ability of clinicians to explore and address competing interests. A recent study describes the development and validation of the Ethical Decision-Making Climate Questionnaire (EDMCQ) [START_REF] Van Den Bulcke | Ethical decision-making climate in the ICU: theoretical framework and validation of a self-assessment tool[END_REF][START_REF] Benoit | Outcome in patients perceived as receiving excessive care across different ethical climates: a prospective study in 68 intensive care units in Europe and the USA[END_REF]. This tool provides the opportunity to develop and evaluate interventions that improve an ICU's ethical climate to facilitate ethical decision-making. Importantly, a clinician's decisions, and the competing interests for those decisions, do not operate in a vacuum, but are instead embedded in a cultural milieu influenced by national policy, financial incentives, resource pressures, patient and family factors, and institutional leadership. A recent qualitative study found that an ICU's ethical priorities influenced the way physicians conceptualized autonomy and beneficence, which consequently influenced communication practices surrounding resuscitation decision-making [START_REF] Dzeng | Influence of institutional culture and policies on do-not-resuscitate decision making at the end of life[END_REF]. The study also revealed the importance of institutional cultural norms that contributed to inappropriately aggressive care at the end of life [START_REF] Dzeng | Homing in on the social: systemlevel influences on overly aggressive treatments at the end of life[END_REF]. Other studies also highlight the understanding of these institutional and ethical norms as an important step to mitigate decisions that are not in the patient's best interests [START_REF] Barnato | Norms of decision making in the ICU: a case study of two academic medical centers at the extremes of end-of-life treatment intensity[END_REF][START_REF] Kelley | Prospective identification of patients at risk for unwarranted variation in treatment[END_REF].

When there are concerns about institutional conflicts of interest, an independent organization, similar to a consumer protection or watch-dog group, could be engaged to perform this monitoring function. Admittedly, hospitals are unlikely to embrace the idea of voluntary data-sharing with a monitoring group. But proactive, voluntary monitoring programs developed with buy-in from hospitals are more likely to set achievable standards than programs created in response to adverse events. As it stands, the task of explaining to the public how competing interests impact their medical care has thus far fallen to journalists. More often than not, these explanations have not been flattering [START_REF] Propublica | God help you. You're on dialysis[END_REF][START_REF] Rivière | Case notes on corruption[END_REF][82][START_REF] Ornstein | At St. Luke's in Houston, Patients Suffer as a Renowned Heart Transplant Program Loses Its Luster[END_REF]. As public trust in political, financial, and criminal justice systems wavers, the medical community would be wise to demonstrate its commitment to a primary goal of protecting patient welfare.

Fig. 2

 2 Fig. 2 Potential role of competing interests in preventing the attainment of patient-centered care and potential tools to address competing interests

Avoidable Describes an acƟon Describes a situaƟon Fig. 1 Conceptual model

	likely to happen in busy hospitals where ICU beds are
	a scarce resource. In these circumstances, patients who
	can safely be treated outside the ICU may be discharged
	or moved to a lower-intensity level of care, even though
	another day of higher-intensity ICU-level care may be
	in that patient's best interest. This situation differs from
	decisions about which ICU patient to treat first because
	the intensivist has entered into a patient-physician rela-
	tionship with the current ICU patient, but not usually
	with the person seeking hospital or ICU admission. Tri-
	age dilemmas are further complicated in rural hospitals
	where the nearest comparable ICU may be many miles
	away. In urban centers with multiple tertiary care cent-
	ers, intensivists arguably have less obligation to admit
	patients if they believe existing ICU patients may be
	adversely affected.

Acknowledgements

The authors wish to thank Medical Librarian Carrie Price, MLS, for her assistance.

Compliance with ethical standards

Author details 1 Division of Pulmonary and Critical Care Medicine, School of Medicine, Johns Hopkins University, 1830 E. Monument St, 5th Floor, Baltimore, MD 21205, USA. 2 Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA. 3 Outcomes After Critical Illness and Surgery (OACIS) Group, Johns Hopkins University, Baltimore, MD, USA. 4 Department for Anesthesiology and Intensive Care, Jena University Hospital, Jena, Germany. 5 Department of Anaesthesiology and Operative Intensive Care Medicine, Charité Universitätsmedizin Berlin, Kreischa, Germany. 6 Patientand Family-Centered Care, Klinik Bavaria, Kreischa, Germany. 7 Interdepartmental Division of Critical Care Medicine, University of Toronto, Toronto, ON, Canada. 8 Department of Critical Care Medicine, Sunnybrook Health Sciences Centre, Toronto, ON, Canada. 9 Intensive Care Unit, Ghent University Hospital, Ghent, Belgium. 10 Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital Saint-Antoine, Service de Réanimation Médicale, Paris, France. 11 Sorbonne Universités, Université Pierre et Marie Curie, Paris, France. 12 Institut National de la Santé et de la Recherche Médicale (INSERM), UMR S 1136, Institut Pierre Louis d'Épidémiologie et de Santé Publique, Paris, France. 13 Department of Intensive Care, Medisch Centrum Leeuwarden, Leeuwarden, The Netherlands. 14 Department of Bioethics and Humanities, University of Washington, Seattle, WA, USA. 15 Division of Pulmonary, Critical Care, and Sleep Medicine, University of Washington, Seattle, WA, USA. 16 Cambia Palliative Care Center of Excellence, University of Washington, Seattle, WA, USA.

Conflicts of interest

The authors declare that no conflict of interest exists.