Cyril Charlier

Guillaume Bouvignies

Philippe Pelupessy

Astrid Walrant

Rodrigue Marquant

Mikhail Kozlov

Pablo De Ioannes

Nicolas Bolik-Coulon

Sandrine Sagan

Patricia Cortes

Nicolas Bolik--Coulon

Aneel K Aggarwal

Ludovic Carlier

Fabien Ferrage

Structure and Dynamics of an Intrinsically Disordered Protein Region That Partially Folds upon Binding by Chemical-Exchange NMR

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Introduction

Intrinsically disordered proteins (IDPs) and regions (IDRs) are ubiquitous in the proteome. 1,2 The flexibility of IDPs allows them to engage in many interactions with other biomolecules. 2 These interactions often lead to folding of a local sequence motif 3 but most parts of a disordered protein usually retain some disorder. In ex-treme cases, an entire IDP assembles in a fuzzy com-plex. 4,5 The presence of both ordered and disordered seg-ments makes the characterization of conformational en-sembles of the bound states of IDPs challenging. 6 A wide-ly applicable method to determine the conformational properties of the interacting regions of complexes involv-ing long IDRs in solution is still missing.

A conventional approach to the study of IDPs has been to identify a minimal peptide that interacts with a folded domain of partner protein and to determine the structure of the "rigid" complex. This reductionist approach has led to numerous structures of complexes between globular domains and peptides, solved by X--ray crystallography or nuclear magnetic resonance (NMR). The analysis of pro-tein--peptide interactions has produced an impressive body of knowledge on the interactions of classes of pro-tein domains with tiny regions of intrinsically disordered proteins, for instance, Src homology 3 (SH3) domains 7,8 or calmodulin. 9,10 Recently, protein structures of folded domains that form a complex with long disordered regions have been explored by X--ray crystallography. Such structures provide little, if any, information on those parts of the IDR that do not become fully ordered in the complex 6,11 while they poten-tially participate in the interaction. NMR has also been used, in particular when most of the disordered region binds to a small domain. 12,13 The characterization of mul-tiple binding sites involving different fragments of the disordered protein is possible. 11,14,15 In rare studies, NMR and crystallography data have been combined to deter-mine the conformational ensemble of a disordered pro-tein bound to a domain. 16,17 This allows one to identify how binding modulates the conformational space of the disordered protein and may reveal secondary transient interactions.

The determination of high--resolution protein structures by NMR remains challenging for large systems (MW > 30 kDa) and this includes the region of a disordered protein that folds upon binding to a large domain. This part of the IDR has comparatively unfavorable relaxation proper-ties, behaving as part of a large domain with slow tum-bling due to the drag of the disordered region. 18,19 Alt-hough perdeuteration and transverse relaxation-optimized methods may attenuate these drawbacks, fast relaxation leads to weak and broad signals that are diffi-cult to identify amongst the many intense and sharp sig-nals from the regions that retain disorder. At worst, con-formational dynamics on µs--ms timescales in the complex may lead to dramatic line broadening. 20--22 Would it be possible to design a general method to de-termine the structure of a complex between an otherwise disordered protein region and a folded protein domain by NMR? This method should be robust enough to be effi-cient even in the absence of exploitable spectra of the complex. Here, we introduce such an approach based on chemical--exchange NMR. Recent progress in the charac-terization of chemical exchange processes by NMR. 23 using Carr--Purcell--Meiboom--Gill (CPMG) relaxation dis-persion (RD) 24,25 and chemical--exchange saturation trans-fer (CEST) 26,27 provide information on weakly populated states that exchange with a ground state easily observed by NMR. CPMG and CEST experiments have been used to determine the kinetics of folding upon binding for some IDRs. 22,28,29 The chemical shifts of a weakly populated state can be obtained from CEST and CPMG and em-ployed as constraints to determine the structure of this NMR--invisible state, whether it is an excited state 30,31 or a complex in exchange with a free state. 32 Weakly populat-ed states with lifetimes between 1 ms and 100 ms are ide-ally suited for these approaches. Interestingly, a signifi-cant share of IDRs form transient but specific complexes with similar lifetimes. 33 We employ CPMG and CEST experiments to characterize the bound form of an intrinsically disordered protein region: a 96--residue long construct from the disordered C--terminal region of the protein Artemis bound to the DNA--binding domain (DBD) of Ligase IV. Artemis is a nuclease, which plays a key role in adaptive immunity and DNA repair through its participation in the non-homologous end--joining (NHEJ) pathway. 34 The structure of a complex between an 11--residue peptide from Artemis and the DBD of Ligase IV has been solved by X--ray crys-tallography. 35,36 The chemical shifts obtained for the bound form of Artemis were used to derive dihedral an-gles for the backbone and a few side chains. We per--formed NMR titrations of the DBD of Ligase IV with Ar-temis to obtain additional structural constraints. We used these data alongside the structure of free Ligase IV to calculate sets of structures with the program HADDOCK. 37 We show that the interaction between Artemis and Ligase IV extends beyond the interface de-fined by X--ray crystallography through additional transi-ent contacts. Overall, we propose an integrative approach based on NMR to characterize both fully and partially ordered regions in the bound state of IDPs.

Results

As a test case, we have investigated the interaction be-tween the disordered C--terminal region of the protein Artemis and the DBD of Ligase IV. Artemis contains a catalytic core comprised of β--lactamase and β--CASP do-mains, as well as a long C--terminal region that is predict-ed to be disordered 38 and proposed to be involved in the regulation of the catalytic activity (see Figure S2.a). We have examined the region of Artemis that encompasses residues 480--575 (Art 480--575 ), which includes residues 485--495, shown previously to interact with the DBD of Ligase IV. 39 The crystal structures of the core of the complex 35,36 (PDB codes 4HTP and 3W1G) are remarkably similar and show the Artemis peptide 485--495 nested in a hydropho-bic pocket between helices α1 and α2 of Ligase IV (Figure S2.b). The ligase IV--binding site of Artemis extends beyond residues 485--495. We obtained a nearly complete NMR assignment of the backbone resonances of Art 480--575 (Fig. S3). The narrow dispersion of the amide 1 H chemical shifts in the 1 H-- 15 N HSQC spectrum shows that Art 480--575 is highly disordered in solution, as confirmed by secondary structure propensities based on chemical shifts 40 (Fig. S13).

An NMR titration shows the extent of the interaction of Art 480--575 with the DBD of Ligase IV ( DBD LigIV). The inten-sities of 1 H--15 N cross--peaks for Artemis residues 485--499 decrease substantially upon the addition of substoichio-metric amounts of unlabeled DBD LigIV (Fig. 1.a), which is a marker of slow to intermediate chemical exchange on the chemical shift timescale. This region includes the 11-residue motif 485--495 that has been originally proposed as the Ligase IV binding site 39 as well as the four following residues at the C--terminus. At stoichiometric amounts of We used isothermal titration calorimetry (ITC) to investi-gate whether the Ligase IV--binding site of Artemis may extend beyond the C--terminus of the previously studied region 485--495. 39 We synthesized and titrated two Arte-mis peptides: Art 485--495 and a longer peptide, Art 485--501 . The dissociation constant for the binding of Art 485--495 to DBD LigIV (K d = 11.7 ± 0.6 µM) is similar to the previously reported value obtained under slightly different condi-tions 35 (Fig. 1.c and Table S1). The addition of six residues at the C--terminus to give Art 485--501 leads to a five--fold re-duction in the dissociation constant (K d = 2.3 ± 0.2 µM), confirming the role of these additional residues in the interaction with DBD LigIV. Since Art 480--575 binds DBD LigIV with identical affinity, residues 502--575 do not contribute significantly to the thermodynamics of the interaction, as suggested by NMR chemical shift perturbations. Under a higher salt concentration (500 mM NaCl) the dissociation constant for the complex between Art 485--495 and DBD LigIV barely changes while the K d for the Art 485--501 : DBD LigIV complex increases 2.6--fold (Table S1, Figure S4). Although all these observations might point to a role of electrostatic interactions involving residues 496--501 of Artemis, the differences in the free energy of binding between Art 485--495 and Art 485--501 arise mostly from a reduction in the entropic penalty upon binding (see discussion in Supporting In-formation).

Characterization of the bound state of Art 480--575 by chemical--exchange NMR. The observation of NMR signals for the core of Art 480--575 in complex with DBD LigIV is challenging at best. CPMG relaxation dispersion and CEST experiments allow the characterization of states that are not visible in NMR spectra, provided that they interconvert with a major, observable state on millisecond time--scales. Analysis of the "invisible" bound form is achieved by analyzing CPMG 28 or CEST 22,41 profiles of resonances from the free protein measured for a sample containing the protein of interest and substoichiometric amounts of the target protein, so that the bound state is lowly populated. We investigated the bound form of Art 480--575 with nitro-gen--15 CPMG relaxation dispersion experiments 42 on samples of 15 N--labeled Art 480--575 with increasing amounts of unlabeled DBD LigIV (molar ratios ranging from 0 to 13 % ) (Fig. 2.a and S5). In the absence of DBD LigIV, only flat profiles are observed and no exchange process can be detected (data not shown). However, addition of merely 3% of DBD LigIV leads to RD profiles showing clear signs of exchange contributions to R 2,eff for 15 N resonances belong-ing to residues 486--499 (Fig. 2.a and S5). Larger RD pro-files are obtained with higher concentrations of DBD LigIV, confirming that the exchange process corresponds to the binding of Art 480--575 to DBD LigIV.

To characterize the complex formation in greater detail, CPMG RD experiments were complemented with 15 N CEST experiments 26,27,43 performed on nitrogen--15 labeled Art 480--575 with 10 % molar ratio of unlabeled DBD LigIV (Fig. 2.b and S6--7). Most sites from the region 486--499 exhibit profiles with two well--separated intensity dips, with the smaller dip corresponding to the bound state resonances, which confirms that, for these sites, 15 N chemical shifts are significantly different between the free and the bound states (Fig. 2.b).

All 15 N CPMG RD and CEST data were simultaneously analyzed using the program ChemEx 44 R 2 ( 15 N) relaxation rates within the interaction region vary drastically upon binding (Figs. 3.a and S14). Higher rates at the core of the interaction site are expected from fold-ing and slow molecular tumbling of DBD LigIV within the complex. Indeed, the average R 2 ( 15 N) rate, over secondary structure elements, measured on a sample of nitrogen--15 labeled DBD LigIV in complex with unlabeled Art 480--575 is <R 2 DBD--bound > = 44 ± 6 s --1 (Fig. S16). R 2 ( 15 N) rates for Arte-mis residues 488--494 range between 45 and 75 s --1 , defin-ing the core of the interface. The highest rates, obtained for residues 491--493, suggest the presence of an additional fast (µs) exchange process in the bound state. By contrast, R 2 ( 15 N) rates confirm the highly disordered nature of re-gions 480--485 and 510--575 in the complex (Fig. 3.a). Inter-estingly, in the bound state, 15 N transverse relaxation R 2 rates for the 495--510 region are lower than in the core of the complex but larger than in the free state. This region spans about twice the persistence length in disordered or unfolded proteins 45 suggesting a partly restricted confor-mational space explored on ps--ns timescales, possibly due to transient interactions with Ligase IV.

Additional structural restraints are necessary to deter-mine the conformation of Art 480--575 in complex with DBD LigIV. Carbon--13 chemical shifts of Art 480--575 in the bound form were obtained from CEST experiments rec-orded on carbonyl, 46 carbon--α, 47 and carbon-β, 47 using a uniformly 15 N, 13 C--labeled sample of Art 480--575 with 10 % molar ratio of unlabeled DBD LigIV (Figs. 2.c--e and S8--10). The large, positive 13 C α secondary shift values 48,49 observed for residues 489--492 in the bound state (Fig. 3.b) strongly suggest these residues form an α--helix in the complex. 50 All the chemical shifts of the bound form of Artemis ob-tained from the combined analysis of CEST profiles were used to derive dihedral backbone angles using the pro-gram Talos--N. 51 These dihedral angles are in excellent agreement with the crystal structures for the core of the complex, except for residues 493 and 494, which are in an extended conformation in solution, but adopt a helical conformation in the crystals (Fig. 3.c). In addition, Talos--N derived dihedral angles χ1 for residues 486 and 491--493. Notably, our approach provides additional structural restraints for residues 495 and 496.

Mapping the binding to Artemis on the surface of Ligase IV. Relevant structural information on the binding surface of Ligase IV may be provided through the use of chemical shifts perturbations upon binding to Artemis. For that purpose, we obtained the site--specific assign-ment of backbone resonances for most of the 240 residues of DBD LigIV (Fig. S15).

The binding surfaces of DBD LigIV to all Artemis constructs were investigated by NMR titrations. Art 480--575 induces selective chemical shift perturbations in the 1 H--15 N TROSY 52 spectrum of DBD LigIV (Fig. S17), showing that binding does not lead to a major conformational rear-rangement in the large globular domain. Residues dis-playing the highest CSP values are mostly localized in the N--terminal region encompassing helices α1 and α2 (Fig. 4.a,c), consistent with the binding interface determined by X--ray crystallography using the Art 485--495 peptide. Sig-nificant perturbations (i.e., one standard deviation be yond the mean CSP) are also observed for residues clus-tered near the loop connecting helices α6 to α7 (138--144), the first half of helix α9 (181--184), and helix α10 (198--206). Apart from residues in helix α10, these regions were sub-stantially less affected when DBD LigIV was titrated by the Art 485--495 peptide, with changes in chemical shifts mostly limited to the N--terminal region (Fig. S18.c). A detailed comparison between the CSP values of Art 480--575 and Art 485--495 (Fig. 4.b,d) suggests a continuous interaction surface that expands beyond the groove between helices α1 and α2, where Art 485--495 lies in the crystal structure. The extension of the surface includes DBD LigIV residues F131, L138, G141, S179, and L181 as well as residues localized at the tip of the hairpin α1--α2 (T22, K28, K30, and G31). Im-portantly, the role of Artemis residues 496--501 in making interactions with these additional docking sites can be inferred from the highly similar CSP profiles obtained with Art 480--575 and the Art 485--501 peptide (Fig. S18.a,b).

NMR data--driven model of the Artemis--Ligase IV complex.

A structural model of the Artemis--Ligase IV complex in solution was determined by docking computa-tion using experimental constraints derived from NMR chemical--exchange and CSPs data. We first determined the structure of Artemis in the bound state by incorporat-ing in CNS 53 the φ, ψ, and χ1 torsion angle restraints de-rived from the chemical shifts of the bound form. To simplify and speed up the analysis, we only considered the binding site 485--501 in the sequence of Artemis (Art 485--501 ) and did not include the region 502--575, which is barely altered by binding. Ten Artemis conformers that are in good agreement with the torsion angle restraints were selected as the starting structural ensemble for the docking process (Table S2). These conformers are re-markably similar to the X--ray structure of Art 485--495 in complex with Ligase IV in the N--terminal part with an average backbone RMSD of 0.62 Å over residues 486--493 (Fig. 5.a). The C--terminal residues 497--501 are highly disordered in the starting NMR ensemble, as expected from the absence of angular constraints for these resi-dues.

HADDOCK is one of the most popular docking programs for the structure determination of biomolecular complex-es using experimental data, in particular NMR CSPs. 37 HADDOCK has been used to determine the structures of homodimers and homo--oligomers of folded proteins from chemical shift perturbations obtained by chemical-exchange NMR 54 as well as the structure of the complex of a short peptide bound to a folded domain 55 based on transferred nuclear Overhauser effects. However, to our knowledge, HADDOCK has never been used in combina-tion with unambiguous structural constraints from chem-ical--exchange NMR to obtain the structure of a complex involving an IDP that folds upon binding. We docked the structures of Artemis in its bound form onto the structure of the free catalytic core of Ligase IV (residues 1--611, PDB 3W5O) 36 using the HADDOCK web server. The "active" and "passive" residues, as defined by HADDOCK to derive ambiguous intermolecular restraints (AIRs), were selected for Ligase IV on the basis of CSPs. The torsion angle re-straints used to calculate the bound structure of Artemis were also used in the docking process. HADDOCK pro-vided 92 final models, sorted in 12 clusters (Fig. S19), each containing 4 to 16 structures with HADDOCK--and Z-scores ranging from -100.7 to -51.4 and from -1.3 to +1.4, respectively. The positions of the N--terminal residues 485--496 of Artemis with respect to Ligase IV are remark-ably similar in the top 3 clusters (Fig. S20). By contrast, the conformations of residues 497--501 and their contacts with Ligase IV differ markedly between the models and even within clusters (Fig. 5.c).

In the top cluster, the average ligand--RMSD upon super-imposition onto DBD LigIV is 2.1 Å for the structured region encompassing Artemis residues 485--496. The structure of the complex with lowest energy is remarkably similar to the high--resolution crystal structure of the Art 485--495 --LigIV complex (Fig. 5.b). The average backbone interface--RMSD with respect to the crystal structure (PDB 3W1G) is 0.9 ± 0.2 Å for the top cluster. The key hydrophobic interac-tions involving P487 and aromatic residues W489, F492, and F493 of Artemis are virtually identical to those ob-tained in the crystal structure. Major differences are only observed for the backbone of residues F493 and K494 that adopt an extended conformation in our NMR structures in solution while they adopt a α--helix conformation in the crystal structures with a short Art 485--495 peptide. The consistency of our approach was evaluated by the comparison of experimental chemical shifts with those predicted with Sparta+ 56 from the 16 structures of the HADDOCK top cluster. Predicted chemical shifts agree very well with experimental shifts (Figure S21) while small differences are observed with the chemical shifts predicted from the crystal structure (pdb code 3W1G), particularly for residues F493 and K494.

Interestingly, the extended conformations of these resi-dues allow the acidic patch that follows towards the C-terminus (including D497, E498, and D501) to face adja-cent regions at the surface of Ligase IV (Fig. 5.c). These additional regions, encompassing the tip of the hairpin α1--α2, the loop α6--α7, and the N--terminal portion of α9, form a continuous positively charged surface. Accord-ingly, the negatively charged residues D497, E498, and D501 of Artemis contact various arginine and lysine residues of Ligase IV in most docking models through electrostatic interactions. The transient nature of these additional contacts is supported by R 2 ( 15 N) rates and chemical shifts which show that Artemis residues 497--501 retain some disorder in the complex. We are well aware that the docking--based structures depicted in Fig. 5.c do not represent a priori the ensemble of confor-mations explored by the Artemis 496--501 region when bound to Ligase IV. However, this ensemble of docking solutions is consistent with the main features identified by NMR data. Predicted chemical shifts from the dock-ing models are in good agreement with experimental values (Fig. S21) and indicate that residues 497--501 are mostly disordered. Weak interactions with the positively charged residues at the surface of Ligase IV may explain the slow decrease of nitrogen--15 R 2 rates in the 497--501 region (Fig. 3.a).

Discussion

The agreement of the NMR--driven docking model with the crystal structure demonstrates that our approach can be applied to obtain structural models of complexes in which the interacting region of an IDP contains both fully ordered and partially disordered regions. The structure of the well--folded parts of the complex com-prising Artemis and Ligase IV only differs from the crys-tal structure, for the backbone dihedral angles of F493 and K494. The proximity of the C--terminus of the short peptide in the crystal structure likely favors the helical conformation. This discrepancy reveals a possible alter-native interpretation of this region in the model of the crystal structure. The C--terminus of the short peptide in the crystal structure is built in a helical conformation. However, high B--factors and a weaker electron density in this region are not inconsistent with a more extended path of this segment as obtained by NMR (Fig. S22). This highlights the importance of using long disordered protein regions to grasp accurately the relevant features of such a complex.

The methodology introduced in this work is particularly well suited to the study of IDPs. Indeed, the bound structures of IDPs are usually limited to isolated sec-ondary structure elements (i.e., extended motifs and alpha helices), which lie against the surface of the folded partner protein. Such structure elements can be (i) well defined with the use of backbone dihedral angle con-straints, and (ii) accurately docked onto the protein partner from the knowledge of its interacting surface. Here, we show that (i) backbone dihedral angles are obtained in a reliable manner through chemical shifts derived from an analysis of CEST profiles and (ii) the interface is sufficiently well characterized by NMR chemical shift perturbations.

A potential limitation of our approach lies in the ability to define the interface of the complex for large folded protein partners. Structural models of complexes be-tween large proteins and peptides or unfolded proteins have been characterized 15 with the use of methyl trans-verse relaxation optimized spectroscopy (methyl TROSY). 57 Protein surfaces contain statistically more methyl--bearing residues at protein--protein interfaces. 58,59 The characterization of protein interac-tions with methyl probes is thus favorable, as was suc-cessfully demonstrated in several studies. 60 CEST pro-files, used to obtain the chemical shifts of the bound form, have been recorded on systems with very high transverse relaxation rates, with R 2 ( 15 N) > 1000 s --1 . 22 This guarantees successful application of our methodology for complexes up to the MDa range.

Our approach is compatible and should now be com-plemented with methods that allow the characterization of the conformational space of IDRs that remain signifi-cantly disordered in the bound state, with the use of programs such as Asteroids 61 or Ensemble. 62 Fragments of the IDR may retain some disorder in the complex and yet be challenging to observe directly by multidimen-sional NMR. In this case, CEST and CPMG experiments can be used to obtain additional information for the determination of conformational ensembles. In particu-lar, CEST and CPMG experiments have been used to obtain a collection of conformational constraints, such as paramagnetic relaxation enhancements, 63 residual dipolar couplings, 64,65 and pseudo--contact shifts 66 of states with a small population in exchange with a major, observable state. Such data would be highly comple-mentary to chemical shifts measured here. Combining our approach for the folded regions of the IDR and these measurements analyzed with Asteroids 61 or Ensemble, 62 will allow the determination of conformational ensem-bles for the entire complex including fully ordered, par-tially ordered, and mostly disordered regions.

Conclusions

Here, we introduce a method for the structural determi-nation of complexes consisting of disordered protein regions that fold in part upon binding to a partner pro-tein. This approach combines chemical--exchange NMR, structural determination of the interacting region of the disordered protein, and docking. Importantly, the method does not require the observation of the bound form of the IDP and provides structural insight on both fully and partially ordered regions in the bound IDP. We apply this method to a complex between a 96--residue disordered region of Artemis and the DBD of Ligase IV. We show that our approach correctly reproduces the crystal structure of the folded core of the complex and provides additional information on regions within the IDP that contribute to the thermodynamic affinity but retain some disorder. The method is not affected by edge effects when short peptides are used in a more traditional approach. From our NMR and ITC--based investigations, we identify additional residues at the C-terminal end of Artemis (496--501) that contribute signif-icantly to the binding to Ligase IV. This NMR--based approach to the structural characterization of complexes of IDPs is robust and is expected to be applicable to a wide range of systems.

Experimental Section

Protein and peptide production. The expression vector for Art 480--575 encodes a 38 kDa fusion protein with an amino-terminal Glutathion S--transferase (GST) domain, a PreScission protease cleavage site, and the Art 480--575 region. Art 480--575 was expressed in E. coli either unlabeled in LB rich medium or uniformly labeled with 15 N or 15 N/ 13 C using the procedure de-veloped by Marley et al.. 67 Art 480--575 was purified by glutathione-affinity chromatography (Genscript) and cleaved on a resin at 4°C with recombinant PreScission protease, followed by size-exclusion chromatography on a Superdex 75 16/60 column (GE Healthcare).

The DNA--binding domain of human Ligase IV ( DBD LigIV; re-gion 1--240) was expressed as a (His)6--tagged fusion protein in a pET--15b vector and purified as previously described. 35 Expres-sion of uniformly labeled 15 N/ 13 C/ 2 D DBD LigIV was carried out from cells cultured in M9 minimum media prepared in 100% D2O. The two Artemis peptides were produced by solid--phase peptide synthesis and purified by RP--HPLC.

Isothermal Titration Calorimetry. ITC experiments were performed on a Nano ITC calorimeter (TA Instruments) at 23 °C. Titrations were carried out with a fixed concentration of DBD LigIV (70--100 µM) into which aliquots of each of the Arte-mis fragments were injected. Thermodynamic parameters were determined by non--linear least--square fitting of the buffer-corrected data using the software NanoAnalyze (version 3.1.2) provided by TA Instruments.

NMR resonance assignments and Chemical Shift Per-turbation (CSP) experiments. NMR spectra were acquired at 296.5 K on Bruker Avance--III HD 600 and 800 MHz spectrom-eters equipped with room--temperature triple resonance ( 1 H, 15 N, 13 C) probes and on a Bruker Avance--III HD 950 MHz spec-trometer equipped with a triple resonance cryoprobe. All NMR data were processed with NMRPipe 68 and analyzed with NMRFAM--Sparky. 69 Backbone resonance assignments of Art 480-- 575 were obtained from the analysis of a series of 3D BEST triple resonances experiments 70 as well as an (H)N(COCA)NH exper-iment, 71,72 all recorded at 800 MHz. The BEST--TROSY versions of these 3D experiments 73 and a 15 N--NOESY--HSQC were rec-orded at 950 MHz to assign the backbone resonances of DBD LigIV. The interaction between Artemis and Ligase IV was probed by chemical shift perturbations (CSP). Uniformly la-beled 15 N--Art 480--575 was titrated at 600 MHz with unlabeled DBD LigIV and followed with 2D 1 H--15 N HSQC spectra. Triple-labeled 2 D/ 15 N/ 13 C--DBD LigIV was titrated with each of the unla-beled Artemis fragments and followed with BEST--TROSY 2D 15 N--1 H correlations at 950 MHz. CSP values were calculated for each residue from the differences in 1 H N (∆𝛿 ! ! ) and 15 N (∆𝛿 ! ) chemical shifts between the free and the bound states using the definition:

𝐶𝑆𝑃 = 𝛥𝛿 ! ! ! + 0.1𝛥𝛿 ! ! CPMG
and CEST experiments. All 15 N CPMG experiments 42 were carried out at a 1 H frequency of 800 MHz on samples of 550 µM 15 N--labeled Art 480--575 with 3%, 6%, 10% and 13% molar ratios of unlabeled DBD LigIV. All CEST experi-ments were acquired at 800 MHz using previously published pulse sequences 26,46,47 on uniformly 15 N--(for 15 N--CEST) and 15 N, 13 C--(for 13 C--CEST) labeled samples of Art 480--575 (550 µM) with 10% molar ratio of unlabeled DBD LigIV. Nitrogen--15 trans-verse relaxation rates (R2) were measured at 800 MHz on a sample containing 300 µM DBD LigIV uniformly labeled with nitrogen--15 in complex with 350 µM unlabeled Art 480--575 .

Analysis of CPMG relaxation dispersion and CEST ex-periments. Peak intensities in CPMG and CEST spectra, 𝐼, were quantified by fitting the lineshape of the peaks using an in--house python script (available upon request). A flowchart in Fig. S1 presents the way the analysis was performed. 15 N CPMG and 15 N CEST data sets were simultaneously fit to a global two-site exchange model between free (F) and bound (B) states of Art 480--575 using the program ChemEx, 44 which simulates the evolution of magnetization during the CPMG and CEST peri-ods by numerically integrating of the Bloch--McConnell equa-tion and minimizes a standard 𝜒 ! target function, as described previously. 26 Fitting parameters include the per residue values 𝜛 ! , 𝑅 2,F , 𝑅 2,B , 𝑅 1 and the global parameter {𝑘 on }, where 𝜛 ! is the chemical shift of the bound state, 𝑅 2,F (𝑅 2,B ) is the trans-verse spin relaxation rate of free (bound) state, 𝑅 1 is the longitudinal spin relaxation rate that is assumed to be the same for both states and 𝑘 on is the association rate constant. The dissociation rate constant, 𝑘 off , was calculated using the relationship 𝑘 off = 𝐾 d 𝑘 on assuming a dissociation constant Kd = 2.2 µM, as determined by ITC experiments. Note that the value of Kd has no influence on the analysis, provided that the binding partner (here DBD LigIV) is saturated (Fig. S11). Indeed, under these conditions the pseudo first--order rate constant is independent of Kd: kon[ DBD LigIV]free = koff α/(1--α), with α the proportion of DBD LigIV added to the solution (in the CEST experiment α = 0.1). 13 C', 13 C α and 13 C β CEST profiles were sub-sequently analyzed using ChemEx employing previously devel-oped protocols. 46,74 Structure calculations and HADDOCK docking. The structure of Art 485--501 bound to Ligase IV was calculated by molecular dynamics with CNS 1.2 in ARIA 2.2, 75 using 26 φ, ψ, and χ1 angular restraints predicted by Talos--N 51 from the back-bone chemical shifts obtained from the analysis of CEST exper-iments. From 30 initial structures refined in explicit water, the 10 lowest energy conformers of Art 485--501 were selected and docked onto the free structure of full--length Ligase IV (PDB 3W5O) on the HADDOCK 2.2 web--server 37 using default pa-rameters. The X--ray structure of full--length Ligase IV in the apo form (PDB 3W5O) and the NMR conformers of Ligase IV-bound Art 485--501 were used as the starting structures. Artemis residues 485--497, for which CEST experiments revealed differ-ences in carbon--13 chemical shifts between the free and the bound states, were considered as directly participating in the binding to Ligase IV and classified as "active" residues. The 26 φ, ψ, and χ1 angular restraints used to calculate the bound structure of Art 485--501 were incorporated into the docking pro-cess as unambiguous constraints. The average ligand--RMSD, as defined following CAPRI standards, 76 was calculated in the top cluster using the program PROFIT. [START_REF] Martin | PROFIT[END_REF] The average backbone interface--RMSD of the docking models to the X--ray structure of the Art 485--495 --Ligase IV complex (PDB 3W1G) was calculated in the top cluster based on interface residues that have a heavy atom within 10 Å of any other interacting partner in the crystal structure.

Figure 1 .

 1 Figure 1. Interaction between Artemis and Ligase IV. (a) Titration of 15 N labeled Art

  single-quantum correlation (HSQC) spectroscopy. (b) Chemical shift perturbations (see methods) between the free and bound forms of Art 480--575 for residues with observable signals in the bound state. (c) Isothermal titration calorimetry of DBD LigIV and three constructs of Artemis: Art 485--495 (green), Art 485--501 (purple), and Art 480--575 (blue).

  DBD LigIV, additional signals can be observed in the [ 1 H--15 N] HSQC spectra of Artemis, which could be assigned to residues 485 and 495--499 in the bound form based on triple resonance experiments. The signals of Artemis residues 486--494 in the bound form remained undetected in both 2D and 3D spectra. The chemical shifts of residues 495--499 drastically change upon Artemis binding to DBD LigIV (Figures 1.a,b). By contrast, the signals of resi-dues flanking the segment 485--499 exhibit rather small chemical shift perturbations (CSP) and are in the fast exchange regime.

Figure 2 .

 2 Figure 2. Chemical--exchange NMR investigation of the bind-ing of Art 480--575 to DBD LigIV. (a) Nitrogen--15 CPMG dispersion profiles for representative residues of Art 480--575 at the inter-face with DBD LigIV. The concentration of DBD LigIV was varied from 0.03 (purple) to 0.06 (orange), 0.10 (green), and 0.13 equivalent (red). (b) Nitrogen--15 CEST profiles obtained for three representative residues of Art 480--575 . Two different rf field amplitudes were used 10.4 Hz (green) and 20.8 Hz (light green). Representative 13 C' (c), 13 C α (d) and 13 C β (e) CEST profiles of residues of Art 480--575 at the interface with DBD LigIV. Experimental data are shown as small circles. Fitted curves are shown as solid lines in all panels. All CEST experiments were recorded in the presence of 0.1 molar equivalent of unlabeled Ligase IV.

  (see Material and Methods) and could be well fit to a global two--site ex-change model: Art 480--575 + LigIV DBD ⇌ Art 480--575 -LigIV DBD with an association rate constant 𝑘 on = 3.00⋅10 7 ± 0.01⋅10 7 s -- 1 .M --1 and a dissociation rate constant 𝑘 off = 𝑘 on • 𝐾 d , = 66.1 ± 0.2 s --1 , where K d = 2.2 µM is the dissociation constant determined by ITC. The analysis also provided nitrogen--15 chemical shifts and effective transverse relaxation rates R 2 ( 15 N) for the bound state of Art 480--575 . Chemical shift differences as large as 7.7 ppm for residue 489 were measured. The nitrogen--15 chemical shifts of residues 486--499 in the bound form are shifted away from random coil values (Fig. S12), clearly indicating that this region becomes more structured when bound to DBD LigIV.

Figure 3 .

 3 Figure 3. Structural and dynamic information on the bound form of Art 480--575 obtained by chemical--exchange NMR. (a) Transverse relaxation rates R 2 ( 15 N) and (b) secondary 13 C a chemical shifts for the free (orange) and bound (green) states of Art 480--575 . (c) Backbone dihedral angles φ (blue) and ψ (red) for the bound state of Art 480--575 . TALOS--N predictions are shown as open symbols, while values found in the crystal structure (PDB 3W1G) are shown as full symbols. Predictions for residue F492 are shown with light colors, as they were not scored the highest confidence level by TALOS--N.

Figure 4 .

 4 Figure 4. Binding surfaces of DBD LigIV to Artemis with chem-ical shift perturbation (CSP). (a) Amide CSP of DBD LigIV induced by the addition of 1 molar equivalent of Art 480--575 . The average CSP value (0.025 ppm) is indicated by a dotted line. (c) Ligase IV residues with a CSP value larger than 0.025 ppm are colored in blue on the X--ray structure of the com-plex DBD LigIV--Art 485--495 (PDB 3W1G) with Artemis in yellow and DBD LigIV in grey. (b) Differences in amide CSP of

Figure 5 .

 5 Figure 5. NMR--based structural models of the complex between Artemis and Ligase IV. (a) Solution structure of Art 485--501 bound to Ligase IV derived from 15 N H , 13 CO, 13 Cα, and 13 Cβ chemical shifts. The 10 NMR conformers of Art 485--501 (yellow) are superim-posed onto residues 486--493 from the crystal structure of Art 485--495 in complex with Ligase IV (orange, PDB 3W1G). (b) Compari-son of the contacts at the interface of the complex in the crystal structure (Artemis in orange and Ligase IV in blue, PDB 3W1G) and in the lowest--energy HADDOCK model of the top cluster (Artemis in yellow and DBD LigIV in green). (c) The 16 models of HADDOCK top cluster are superimposed onto the crystal structure of the complex as in panel (b). The electrostatic surface potential map of DBD LigIV is shown in blue, white, and red to indicate positive, neutral, and negative electrostatic surface poten-tials, respectively. Residues of Artemis with acidic side--chains (D497, E498, and D501) are represented by small red spheres.

Table of

 of Contents graphic.

ACKNOWLEDGMENT

The authors would like to thank Patrick Fuchs (Université Paris Diderot), Damien Laage (Ecole normale superieure) and Guillaume Stirnemann (Institut de Biologie Physico--Chimique) for fruitful discussions as well as Kaushik Dutta (New York Structural Biology Center), Adrien Favier (Insti-tut de Biologie Structurale, Grenoble) for assistance with NMR experiments, and Geoffrey Bodenhausen (Ecole Nor-male Supérieure) for his careful reading of the manuscript.

ABBREVIATIONS

CEST, chemical--exchange saturation transfer; CPMG, Carr--Purcell--Meiboom--Gill; CSP, chemical shift perturbations; DBD, DNA--binding domain; GST, Glutathion S--transferase; IDP, intrinsically disordered protein; IDR, intrinsically disordered region; ITC, isothermal titration calorimetry; NHEJ, non--homologous end--joining; NMR, nuclear mag-netic resonance; RD, relaxation dispersion; SH3, Src ho-mology 3.

Funding Sources

This research has received funding from the European Research Council (ERC) under the European Community's Seventh Framework Programme (FP7/2007--2013), ERC Grant agreement 279519 (2F4BIODYN) to F.F. as well as the Equipex contract ANR--10--EQPX--09. Financial support from the TGIR--RMN--THC FR 3050 CNRS is gratefully acknowl-edged. A. K. A. is a member of the New York Structural Biology Center, which is a STAR center supported by the New York State Office of Science, Technology, and Aca-demic Research.

ASSOCIATED CONTENT

Supporting Information

Extended experimental section; prediction of disorder; ITC parameters; assigned 2D correlations of Art ; full docking results. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION

Corresponding Authors * Ludovic.Carlier@upmc.fr; Fabien.Ferrage@ens.fr Present Addresses † Laboratory of Chemical Physics, NIDDK, NIH, Bethesda, MD, 20892, USA

Author Contributions

The manuscript was written through contributions of all authors.


David Lebeaux
email: david.lebeaux@yahoo.fr

Emmanuelle Bergeron

Jérémy Berthet

Juliette Djadi-Prat

Delphine Mouniée

Patrick Boiron

Olivier Lortholary

Véronica Rodriguez-Nava
email: rodriguez-nava@univ-lyon1.fr

Antibiotic susceptibility testing and species identification of Nocardia isolates: a retrospective analysis of data from a French expert laboratory, 2010-2015

Keywords: Nocardia, disk diffusion, 16S, hsp65

Objectives. Nocardia, a Gram-positive bacterium, is responsible for rare and severe infections. Accurate microbiological data are essential to guide antibiotic treatment. Our primary objective was to describe species identification and results of antimicrobial susceptibility testing (AST) for Nocardia isolates analysed over a 6year period. Secondary objectives were to study temporal trends in species distribution and AST results.

Methods. We retrospectively analysed results from Nocardia isolates sent between January 2010 and December 2015 to a French laboratory dedicated to Nocardia (Observatoire Français des Nocardioses). Species identification was obtained by amplification and sequencing of a 600bp fragment of the 16S rRNA gene (for all isolates) and of hsp65 (when required). AST was performed using disk diffusion.

Results. We included 793 Nocardia isolates, mostly from the lungs (53.8%). The most frequent species were N. farcinica (20.2%), N. abscessus complex (19.9%) and N. nova complex (19.5%). The proportion of N. farcinica increased significantly over time from 13% in 2010 to 27.6% in 2014. Linezolid, amikacin, trimethoprimsulfamethoxazole, minocycline, and imipenem were the most frequently identified active antibiotics with, respectively, 0% (0/734), 2.9% (21/730), 5.4% (40/734), 9.4% (69/734) and 19.5% (143/732) of isolates not susceptible. N. farcinica was frequently not susceptible to cefotaxime (118/148, 79.7% of the isolates), but only about 5% of N. cyriacigeorgica and N. abscessus complex isolates were not susceptible to cefotaxime.

Conclusions.

In this first epidemiological study of Nocardia isolated from human samples in France, N. farcinica was the species most frequently identified and its prevalence increased over time.

INTRODUCTION

Nocardia species are Gram-positive filamentous bacteria found in a wide range of natural environments including decaying vegetation, soil and water [START_REF] Brown-Elliott | Clinical and laboratory features of the Nocardia spp. based on current molecular taxonomy[END_REF][START_REF] Saubolle | Nocardiosis: review of clinical and laboratory experience[END_REF]. The use of molecular microbiology, including amplification and sequencing of the 16S RNA, hsp65 and other genes, has led to the description of more than 100 species, so far [START_REF] Brown-Elliott | Clinical and laboratory features of the Nocardia spp. based on current molecular taxonomy[END_REF][START_REF] Euzeby | List of Bacterial Names with Standing in Nomenclature: a folder available on the Internet (http://www.bacterio.net[END_REF]. Nocardia can be responsible for severe opportunistic infections in immunocompromised patients and patients with chronic bronchopulmonary diseases [START_REF] Brown-Elliott | Clinical and laboratory features of the Nocardia spp. based on current molecular taxonomy[END_REF][START_REF] Coussement | Nocardia infection in solid organ transplant recipients: a multicenter European case-control study[END_REF][START_REF] Minero | Nocardiosis at the turn of the century[END_REF][START_REF] Rodriguez-Nava | A French multicentric study and review of pulmonary Nocardia spp. in cystic fibrosis patients[END_REF]. Infection follows bacterial inhalation or, less frequently, direct inoculation through the skin and can lead to pneumonia, brain abscesses and/or skin/softtissue infections [START_REF] Brown-Elliott | Clinical and laboratory features of the Nocardia spp. based on current molecular taxonomy[END_REF].

The ideal initial treatment for nocardiosis should cover a broad-range of species with adequate antibiotic concentration in all involved organs [START_REF] Lebeaux | Nocardiosis in transplant recipients[END_REF]. However, defining the optimal treatment is difficult because of the lack of comparative and prospective clinical data. As a consequence, most antibiotic regimens currently proposed rely on microbiological data, including species identification and antimicrobial susceptibility testing (AST) [START_REF] Larruskain | Susceptibility of 186 Nocardia sp. isolates to 20 antimicrobial agents[END_REF][START_REF] Schlaberg | Susceptibility profiles of Nocardia isolates based on current taxonomy[END_REF][START_REF] Valdezate | Epidemiology and susceptibility to antimicrobial agents of the main Nocardia species in Spain[END_REF]. Notably, each Nocardia species has a specific antibiotic susceptibility pattern [START_REF] Larruskain | Susceptibility of 186 Nocardia sp. isolates to 20 antimicrobial agents[END_REF][START_REF] Schlaberg | Susceptibility profiles of Nocardia isolates based on current taxonomy[END_REF][START_REF] Valdezate | Epidemiology and susceptibility to antimicrobial agents of the main Nocardia species in Spain[END_REF][START_REF] Wallace | Antimicrobial susceptibility patterns of Nocardia asteroides[END_REF]. Obtaining reliable species identification using molecular methods is therefore essential to guide initial antibiotic treatment.

We retrospectively collected data from a 6-year period (2010-2015) from a French laboratory dedicated to Nocardia (Observatoire Français des Nocardioses [OFN]). Our primary objective was to describe species distribution and AST results. Secondary objectives were to study temporal trends in species distribution, AST results and geographic distribution of Nocardia in France over the 6-year period.

METHODS

Study design, settings and inclusion criteria

We retrospectively reviewed results for isolates sent to the OFN for Nocardia testing between January 2010 and December 2015. The OFN is a French laboratory that has specialised in Nocardia and other Actinomycetes since 1999 [START_REF] Boiron | Review of nocardial infections in France 1987 to 1990[END_REF]. French microbiology laboratories (from continental France and overseas territories) can send biological samples or bacterial isolates for molecular identification and/or AST; isolates from neighbouring countries are also analysed. The isolate density for each region of France was defined as the number of Nocardia isolates sent to our centre per year for 100,000 population of that region; population data for 2016 were used, obtained from the Institut national de la statistique et des études économiques (INSEE) [13].

Isolates were included in the present study if they fulfilled the following criteria: i) molecular-based confirmation that the strain belonged to the genus Nocardia (see below); ii) the strain had been isolated from a human clinical sample; iii) species identification had been performed by molecular biology (see below).

Variables

The following variables were recorded: demographic data (age and patient sex), year and geographic location of the isolated strain, type of clinical sample from which the Nocardia strain had been isolated (sputum, bronchial aspirate, bronchoalveolar lavage, pleural fluid, protected-specimen brush, cerebrospinal fluid, abscess fluid, organ biopsy, blood cultures), species identified and need to amplify hsp65, results of the AST.

Continuous variables are presented as means (± standard deviation) or medians (range). Categorical variables are presented as numbers and frequencies.

Microbiology

Since 1999, the OFN uses a standardised protocol for microbiological analyses of Nocardia: i) confirmation that the bacterial strain belongs to the genus Nocardia using a 16S-based Nocardia polymerase chain reaction (PCR) performed directly on the bacterial colony on an agar plate [START_REF] Couble | Direct detection of Nocardia spp. in clinical samples by a rapid molecular method[END_REF];

ii) species identification obtained by amplification and sequencing of a fragment of ~600 base pairs (bp) of the gene coding for the 16S ribosomal RNA (16S rRNA) using PCR [START_REF] Cloud | Evaluation of partial 16S ribosomal DNA sequencing for identification of Nocardia species by using the MicroSeq 500 system with an expanded database[END_REF]. The sequence obtained in this second step is compared to those stored in GenBank using blast alignment software (http://www.ncbi.nlm.nih.gov/blast) and the BIBI (Bio Informatic Bacteria Identification tool: https://umr5558-bibiserv.univ-lyon1.fr/lebibi/lebibi.cgi) [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF]. Identification at the species level requires 99.6% sequence similarity with the type strain of a single species. If more than 1 sequence in the database has more than 99% similarity, identification is made at the level of the complex. For N. abscessus complex or N. transvalensis complex, a 440 bp fragment of the hsp65 gene is amplified and sequenced to obtain species identification [START_REF] Rodriguez-Nava | Use of PCR-restriction enzyme pattern analysis and sequencing database for hsp65 gene-based identification of Nocardia species[END_REF]. Isolates with sequence similarities < 99% are identified as Nocardia spp.. iii) AST is performed using the disk diffusion method on cation-adjusted Mueller-Hinton (CA-MH) agar plates.

Inoculums are prepared according to the Clinical and Laboratory Standards Institute (CLSI) standard M24-A2 [START_REF] Clsi | Susceptibility Testing of Mycobacteria, Nocardiae and Other Aerobic Actinomycetes; Approved Standard-Second Edition[END_REF][START_REF] Ambaye | Comparison of agar dilution, broth microdilution, disk diffusion, E-test, and BACTEC radiometric methods for antimicrobial susceptibility testing of clinical isolates of the Nocardia asteroides complex[END_REF]. The antibiotic disks used are described in Supplementary Table 1. Results are read after 72 h of culture. For each antibiotic disk, the diameter of the inhibition zone is recorded and compared to thresholds For trimethoprim-sulfamethoxazole, the plates are read at 80% of growth inhibition. If the inhibition zone is < 10 mm, an E-test strip is performed on a CA-MH agar plate [START_REF] Lowman | Antimicrobial susceptibility testing and profiling of Nocardia species and other aerobic actinomycetes from South Africa: comparative evaluation of broth microdilution versus the Etest[END_REF]. If the trimethoprim-sulfamethoxazole minimum inhibitory concentration (MIC) obtained on the E-test strip is ≤ 2/38 µg/mL, the isolate is considered susceptible; if the MIC is ≥ 4/76 µg/mL, the strain is considered resistant [START_REF] Clsi | Susceptibility Testing of Mycobacteria, Nocardiae and Other Aerobic Actinomycetes; Approved Standard-Second Edition[END_REF].

(

Ethical aspects

This study was approved by the Comité de Protection des Personnes (CPP) Ile-de-France I Ethical board (CPPIDF1-2015-octobre-DAP 33), the CCTIRS (Comité consultatif sur le traitement de l'information en matière de recherche dans le domaine de la santé, file 16-355 approved May 19, 2016) and the CNIL (Comité National Informatique et Liberté).

RESULTS

Description of the isolates collected

During the study period (2010-2015), 793/823 Nocardia isolates met our entry criteria and were included in the analysis. Most isolates originated from France (696/793, 88%) (Table 1). The median patient age at sampling was 66 years. Most of the Nocardia isolates were isolated from the lungs (427/793, 53.8%) or from subcutaneous abscesses or skin (156/793, 19.7%) (Table 1).

The most frequent species identified were N. farcinica (20.2%), N. abscessus complex (19.9%), N. nova complex (19.5%) and N. cyriacigeorgica (12.9%) (Table 2). N. farcinica was the most frequently isolated species in blood cultures and brain abscesses/cerebrospinal fluid: 21/39 (54%) and 19/43 (44.2%), respectively (Supplementary table 2). To obtain species identification for the N. abscessus and N. transvalensis complexes, hsp65 amplification and sequencing was required for 179/793 isolates (22.6%).

Antibiotic susceptibility test results

AST was performed for 736 of the Nocardia isolates (92.8%). Linezolid, amikacin, trimethoprimsulfamethoxazole, minocycline and imipenem were the antibiotics most frequently identified as being active against Nocardia with, respectively, 0%, 2.9%, 5.4%, 9.4% and 19.5% of isolates being non-susceptible (Table 3). For the N. abscessus complex, cefotaxime and ceftriaxone were the β-lactam antibiotics most frequently identified as being active (less than 3% of isolates were not susceptible), followed by meropenem and imipenem (7.3 and 11.8% of non-susceptible isolates, respectively) (Table 3). Conversely, N. farcinica isolates were frequently not susceptible to cefotaxime and ceftriaxone (~80% of the isolates) and meropenem (73% of isolates) but were frequently susceptible to amoxicillin/clavulanic acid or imipenem, with 20.1% and 23% of non-susceptible isolates, respectively. N. nova was more frequently not susceptible to amoxicillin-clavulanate (91.6%) than to amoxicillin (23.1%) and was infrequently not susceptible to imipenem (0.7%) and cefotaxime (20.3%). N. cyriacigeorgica was frequently not susceptible to amoxicillin-clavulanate (90.5%) and amoxicillin (87.2%) but was infrequently not susceptible to cefotaxime (7.4%) and imipenem (10.5%). N. brasiliensis was frequently not susceptible to amoxicillin (66.7%) and imipenem (85.1%) and amoxicillin-clavulanate was the most frequently active β-lactam (8.3% of N. brasiliensis isolates were not susceptible).

For carbapenem antibiotics, N. farcinica and N. cyriacigeorgica isolates were more frequently not susceptible to meropenem than to imipenem. Conversely, N. transvalensis complex and N. brasiliensis were more frequently not susceptible to imipenem than to meropenem. Non-susceptibility to different carbapenems was equally frequent for N. abscessus and N. nova complexes.

Less than 3% of the isolates were not susceptible to amikacin, with the notable exception of N. transvalensis complex in which 30.6% (15/49) were not susceptible. For gentamicin and tobramycin, more than 90% of N. farcinica and N. transvalensis complex isolates were not susceptible; 80% of N. nova complex isolates were not susceptible to tobramycin (Table 3).

Less than 10% of isolates were not susceptible to minocycline, but some species had higher rates of nonsusceptibility, e.g., N. cyriacigeorgica (18.9%), N. farcinica (12.8%) and N. brasiliensis (12.5%).

Temporal trends

Between 2010 and 2013, the proportion of isolates identified as N. farcinica increased from 13% to 28.0%, decreasing thereafter to 19.3% in 2015 (overall Chi-square test, p=0.038) (Figure 1).

The frequency of isolates that were not susceptible to amikacin or trimethoprim-sulfamethoxazole remained stable between 2010 and 2015 at less than 9% for both drugs (Supplementary Figure 1).

Geographic distribution of Nocardia species

Among Nocardia isolates from France, isolate density was greater in Occitanie (the southernmost region of continental France), New Caledonia, Reunion and Guadeloupe than in other regions, with densities of 0.46, 0.42, 0.39 and 0.29 isolates/year/100,000 population, respectively (Figure 2). In continental France, isolate density was greater in the south east of the country than in other areas.

DISCUSSION

In this retrospective study, we analysed data from a French microbiology laboratory dedicated to the study of aerobic Actinomycetes. Among the 793 Nocardia isolates included, N. farcinica, N. abscessus complex, N. nova complex and N. cyriacigeorgica were the most frequently identified species, representing about three quarters of all isolates. In our sample, linezolid, amikacin and trimethoprim-sulfamethoxazole were the antibiotics that were most frequently identified as being active, each with less than 9% of isolates being non-susceptible.

The distribution of species we observed was different from that reported in recent studies from Spain (1119 isolates) and the United States of America (1299 isolates) in which N. cyriacigeorgica and N. nova complex were most frequently identified [START_REF] Schlaberg | Susceptibility profiles of Nocardia isolates based on current taxonomy[END_REF][START_REF] Valdezate | Epidemiology and susceptibility to antimicrobial agents of the main Nocardia species in Spain[END_REF]. Because a 16S rRNA +/-hsp65 molecular identification strategy was used in Spain and in the present report, it is unlikely that species misclassification could explain these discrepancies. Of note, our isolates of N. farcinica, the most frequently identified species in our study, were not part of an outbreak. One possible explanation for the differences in species distribution is that climatic conditions may have an impact on the epidemiology of Nocardia, as suggested by the observation that N. brasiliensis is predominantly isolated in tropical or subtropical regions [START_REF] Brown-Elliott | Clinical and laboratory features of the Nocardia spp. based on current molecular taxonomy[END_REF]. Furthermore, higher isolate density was observed in the South of France and overseas territories than in other regions, suggesting that environmental conditions may also increase the spread of Nocardia. The high densities observed in Reunion and New Caledonia also raise the possibility of an underlying genetic condition that may favour this opportunistic condition, such as alveolar proteinosis [START_REF] Hadchouel | Biallelic Mutations of Methionyl-tRNA Synthetase Cause a Specific Type of Pulmonary Alveolar Proteinosis Prevalent on Reunion Island[END_REF].

A phylogenetic analysis revealed that the 16S rRNA gene was not sufficient to obtain precise identification for some Nocardia complexes, such as abscessus or transvalensis [START_REF] Mctaggart | Phylogeny and identification of Nocardia species on the basis of multilocus sequence analysis[END_REF]. Conversely, the 16S rRNA gene had sufficient discriminatory power for isolates belonging to the N. nova complex, N. farcinica, N. cyriacigeorgica or brasiliensis species [START_REF] Schlaberg | Susceptibility profiles of Nocardia isolates based on current taxonomy[END_REF]. Although our pragmatic approach (16S rRNA +/-hsp65) appears attractive for routine purposes, recent studies have demonstrated that the analysis of concatenated sequences of several genes, such as multilocus sequence analysis (MLSA), is likely to be more reliable for phylogenetic purposes [START_REF] Mctaggart | Phylogeny and identification of Nocardia species on the basis of multilocus sequence analysis[END_REF]. In the near future, whole genome sequencing may also help to decipher Nocardia phylogeny.

Among the 736 Nocardia isolates that underwent AST, the antibiotics most frequently identified as active against Nocardia were linezolid, amikacin, trimethoprim-sulfamethoxazole, minocycline and imipenem.

The main challenge in the field of Nocardia AST is the lack of data correlating AST results with clinical outcomes. As a consequence, technical guidelines are derived from in vitro studies assessing the reproducibility and the technical pitfalls of each method. In our study, AST was performed by disk diffusion, even though the CLSI has stated that broth microdilution is the technical gold-standard for Nocardia [START_REF] Clsi | Susceptibility Testing of Mycobacteria, Nocardiae and Other Aerobic Actinomycetes; Approved Standard-Second Edition[END_REF]. However, the CLSI also acknowledged that broth microdilution may have limitations, including false-resistant results for ceftriaxone against N. brasiliensis or for imipenem against N. farcinica [START_REF] Clsi | Susceptibility Testing of Mycobacteria, Nocardiae and Other Aerobic Actinomycetes; Approved Standard-Second Edition[END_REF]. Sulphonamide testing with broth microdilution is also challenging and requires disk diffusion for confirmation if a resistant strain is identified [START_REF] Clsi | Susceptibility Testing of Mycobacteria, Nocardiae and Other Aerobic Actinomycetes; Approved Standard-Second Edition[END_REF]. Another pitfall of broth microdilution is its poor interlaboratory reproducibility, especially for some antibiotic/species combinations, such as ceftriaxone against N. cyriacigeorgica and N. wallacei or sulphonamides against N. farcinica and N. wallacei [START_REF] Conville | Multisite reproducibility of the broth microdilution method for susceptibility testing of Nocardia species[END_REF]. Furthermore, testing several agents with broth microdilution is time-consuming, especially given the lack of available commercial plates dedicated to Nocardia testing in Europe. As a consequence, alternative methods are required to routinely perform AST for Nocardia. First described in 1973 by Michael C. Bach and co-workers, antibiotic disk diffusion was further developed and analysed by Richard Wallace's group [START_REF] Bach | Activity of minocycline against Nocardia asteroides: comparison with tetracycline in agar-dilution and standard disc-diffusion tests and with sulfadiazine in an experimental infection of mice[END_REF][START_REF] Wallace | Disk diffusion susceptibility testing of Nocardia species[END_REF]. In 1997, results from a direct comparison of broth microdilution, antibiotic disk diffusion, agar dilution, E-test and the BACTEC radiometric method on 26 Nocardia isolates were published [START_REF] Ambaye | Comparison of agar dilution, broth microdilution, disk diffusion, E-test, and BACTEC radiometric methods for antimicrobial susceptibility testing of clinical isolates of the Nocardia asteroides complex[END_REF]. There was 100% agreement of antibiotic disk diffusion results with the study "gold standard" for amikacin, erythromycin, imipenem, minocycline and trimethoprim-sulfamethoxazole; conversely, 80 to 88% agreement was observed for ampicillin, amoxicillin-clavulanic acid, ceftriaxone and ciprofloxacin. Overall agreement for the disk diffusion method was 95.7%. Of note, this study included only a small number (9/27) of the antibiotics we tested. The discrepancies regarding β-lactam results may be related to inducible β-lactamase whose production can vary depending on the experimental conditions. The comparison of results from large-scale microbiological studies performed with broth microdilution [START_REF] Larruskain | Susceptibility of 186 Nocardia sp. isolates to 20 antimicrobial agents[END_REF][START_REF] Schlaberg | Susceptibility profiles of Nocardia isolates based on current taxonomy[END_REF], E-tests [START_REF] Valdezate | Epidemiology and susceptibility to antimicrobial agents of the main Nocardia species in Spain[END_REF] and disk diffusion (present study) highlights the difficulties in interpreting Nocardia AST results (see a comprehensive overview of these data on Supplementary table 3) [START_REF] Larruskain | Susceptibility of 186 Nocardia sp. isolates to 20 antimicrobial agents[END_REF][START_REF] Schlaberg | Susceptibility profiles of Nocardia isolates based on current taxonomy[END_REF][START_REF] Valdezate | Epidemiology and susceptibility to antimicrobial agents of the main Nocardia species in Spain[END_REF]. Our results using disk diffusion to assess susceptibility of cefotaxime/ceftriaxone against N. cyriacigeorgica (low frequency of resistance) and against N. farcinica (high frequency of resistance) were similar to those from studies using other methods [START_REF] Larruskain | Susceptibility of 186 Nocardia sp. isolates to 20 antimicrobial agents[END_REF][START_REF] Schlaberg | Susceptibility profiles of Nocardia isolates based on current taxonomy[END_REF][START_REF] Valdezate | Epidemiology and susceptibility to antimicrobial agents of the main Nocardia species in Spain[END_REF]. Conversely, there were significant discrepancies regarding imipenem susceptibility against N. farcinica with 67% of isolates identified as resistant by broth microdilution [START_REF] Schlaberg | Susceptibility profiles of Nocardia isolates based on current taxonomy[END_REF], compared to 4% by E-test [START_REF] Valdezate | Epidemiology and susceptibility to antimicrobial agents of the main Nocardia species in Spain[END_REF] and 23% by disk diffusion. These results raise the question of stability issues for imipenem in broth microdilution and stress the need to interpret AST results according to the method used [START_REF] Viaene | Comparative stability studies of antipseudomonal beta-lactams for potential administration through portable elastomeric pumps (home therapy for cystic fibrosis patients) and motor-operated syringes (intensive care units)[END_REF].

To our knowledge, this study is the first in which Nocardia epidemiology has been assessed over time and across geographical regions in France. The proportions of most species remained stable over time with the notable exception of N. farcinica for which the proportion increased significantly between 2010 and 2014. We recently observed that N. farcinica was more common among solid organ transplant (SOT) recipients [START_REF] Coussement | Nocardia infection in solid organ transplant recipients: a multicenter European case-control study[END_REF][START_REF] Lebeaux | Outcome and Treatment of Nocardiosis After Solid Organ Transplantation: New Insights From a European Study[END_REF]. As SOT now appears to be one of the leading and increasingly common conditions favouring nocardiosis, this increase in the proportion of N. farcinica may be explained by an increase in the proportion of cases of post-SOT nocardiosis.

The main strengths of our study include the large number of isolates reliably identified at the species level using standardised methods performed by the same technical team with the same guidelines during the study period. However, there are also several limitations, including the fact that reporting nocardiosis is not mandatory in France. Thus, the isolates sent to the OFN may reflect a particular interest of the local clinical microbiology laboratory or a lack of knowledge in this field. Nevertheless, although we have described the largest sample of Nocardia isolates in France so far, it is likely that we missed some cases.

In conclusion, we provide results from the first large-scale epidemiological study of Nocardia in France.

N. farcinica was the most frequently identified species and identification rates increased over time. AST performed by disk diffusion appears to be a reliable method for routine testing of Nocardia. the number of isolates collected during the study period and then the "isolate density" 10 defined as the number of isolates per year for 100,000 population. The colour code 11 represents the number of isolates/year/100,000 population for each region. One isolate for 12

TABLES AND FIGURES

Mayotte Island and one isolate for French Polynesia are not depicted. Occitanie is the 13 southernmost region of continental France and is coloured in black. 14

FIGURES 1 Figure 1 . 6 7Figure 2 . 8 (

 11628 FIGURES 1

Supplementary Table 1) [20]. A

  "non-susceptible" isolate is defined as being resistant or intermediate.

	Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Nocardia asteroides ATCC 19247T were
	used as quality control organisms.

Table 1 . Characteristics of 793 Nocardia isolates analysed at the Observatoire Français des Nocardioses (2010-2015)

 1

		793
	Characteristics	Nocardia
		isolates
	Patient demographical data
	Age at Nocardia sampling (years) (median, range) n= 693	66 [4-90]
	Male (n, %) n=743	432 (58.1)
	Year of sampling (n, %)
	2010	123 (15.5)
	2011	130 (16.4)
	2012	127 (16.0)
	2013	132 (16.6)
	2014	141 (17.8)
	2015	140 (17.7)
	Country of origin (n, %) n=791
	France	696 (88.0)
	Belgium	31 (3.9)
	Switzerland	28 (3.5)
	Netherlands	23 (2.9)
	Portugal	7 (0.9)
	Spain	2 (0.3)
	Monaco	1 (0.1)
	Lebanon	1 (0.1)
	Luxembourg	1 (0.1)
	Tunisia	1 (0.1)
	Site and method of sampling (n, %)
	Lung	427 (53.8)
	Sputum	156 (19.7)
	Bronchoalveolar lavage	112 (14.1)
	Bronchial aspirate	99 (12.5)
	Lung biopsy	20 (2.5)
	Pleural fluid	20 (2.5)
	Bronchial biopsy	1 (0.1)
	Protected-specimen brush	1 (0.1)
	Unspecified	18 (2.3)
	Cutaneous biopsy or subcutaneous abscess sample	156 (19.7)
	Blood culture	39 (4.9)
	Brain abscess	37 (4.7)
	Joint fluid	18 (2.3)

Table 2 . Identified species for the 793 Nocardia isolates.

 2

		793
	Nocardia species	Nocardia
		isolates
	N. farcinica	160 (20.2)
	N. abscessus complex	158 (19.9)
	N. abscessus sensu stricto	59
	N. abscessus/N.araoensis-like	15
	N. abscessus/N.arthritidis-like	6
	N. arthritidis	9
	N. arthritidis/gamkensis/exalbida-like	30
	N. beijingensis	22
	N. asiatica	4
	N. testacae	2
	N. abscessus complex	11
	N. nova complex	155 (19.5)
	N. nova sensu stricto	144
	N. veterana	11
	N. cyriacigeorgica	102 (12.9)
	N. brasiliensis	55 (6.9)
	N. transvalensis complex	51 (6.4)
	N. wallacei	39
	N. transvalensis sensu stricto	11
	N. blacklockiae	1
	N. otitidiscaviarum	13 (1.6)
	N. brevicatena/paucivorans complex	11 (1.4)
	N. pseudobrasiliensis	8 (1.0)
	N. flavorosea	7 (0.9)
	N. cerradoensis	6 (0.8)
	N. mexicana	4 (0.5)
	N. carnea	3 (0.4)
	N. jiangxiensis	3 (0.4)
	N. goodfellowii	2 (0.3)
	N. asteroides	2 (0.3)
	N. puris	2 (0.3)
	N. higoensis	2 (0.3)
	N. mikamii	2 (0.3)
	N. pneumoniae	2 (0.3)
	N. coubleae	1 (0.1)
	N. altamirensis	1 (0.1)
	N. elegans	1 (0.1)
	N. neocaledoniensis	1 (0.1)

Table 3 . Results of antibiotic susceptibility testing among 736 Nocardia isolates according to species

 3 Non-susceptible trains were defined as resistant or intermediate. Other species who underwent AST were: N. otitidiscaviarum (n=11), N. brevicatena/paucivorans complex (n=9), N. pseudobrasiliensis (n=8), N. flavorosea (n=7), N. cerradoensis (n=4), N. mexicana (n=4), N. carnea (n=3), N. jiangxiensis (n=2), N. asteroides (n=2), N. higoensis (n=2), N. mikamii (n=2), N. pneumoniae (n=2), N. goodfellowii (n=1), N. puris (n=1), N. coubleae (n=1), N. altamirensis (n=1), N. elegans (n=1), N. neocaledoniensis (n=1), N. concava (n=1), N. rhamnosiphila (n=1), N. takedensis (n=1), N. uniformis (n=1), N. vinacea (n=1), Nocardia spp. (n=31).

	Antibiotic, n (%) of non-susceptible* isolates	All Nocardia isolates. (n=736)	Nocardia abscessus complex (n=152)	Nocardia farcinica (n=149)	Nocardia nova complex (n=145)	Nocardia cyriacigeorgica (n=95)	Nocardia transvalensis complex (n=49)	Nocardia brasiliensis (n=48)
	Amoxicillin	394 (53.8)	37 (24.3)	137 (91.9)	33 (23.1)	82 (87.2)	29 (59.2)	32 (66.7)
	Amoxicillin-clavulanic acid 365 (49.7)	40 (26.3)	30 (20.1)	131 (91.6)	86 (90.5)	6 (12.2)	4 (8.3)
	Ticarcillin-clavulanic acid	372 (53.8)	42 (27.6)	58 (43.6)	131 (94.2)	82 (93.2)	5 (10.9)	5 (10.9)
	Piperacillin-tazobactam	457 (66.0)	39 (26.7)	129 (97.0) 103 (74.1)	83 (93.3)	31 (67.4)	17 (37.0)
	Imipenem	143 (19.5)	18 (11.8)	34 (23.0)	1 (0.7)	10 (10.5)	18 (36.7)	40 (85.1)
	Meropenem	227 (31.2)	11 (7.3)	108 (73.0)	6 (4.2)	41 (43.6)	8 (16.7)	28 (58.3)
	Doripenem	119 (29.1)	8 (9.4)	68 (68.7)	4 (5.1)	11 (25.0)	2 (6.9)	16 (76.2)
	Ertapenem	303 (42.7)	17 (11.6)	123 (89.8)	18 (12.7)	59 (62.8)	23 (48.9)	31 (64.6)
	Cefotaxime	193 (26.4)	4 (2.7)	118 (79.7)	29 (20.3)	7 (7.4)	0 (0.0)	11 (22.9)
	Ceftriaxone	209 (28.5)	4 (2.6)	120 (80.5)	42 (29.4)	4 (4.2)	2 (4.1)	15 (31.3)
	Cefepime	281 (38.9)	12 (7.9)	134 (91.2)	33 (23.4)	33 (35.1)	8 (17.0)	27 (56.3)
	Cefuroxime	98 (25.0)	3 (3.8)	64 (69.6)	7 (9.1)	1 (2.2)	1 (3.6)	9 (42.9)
	Pristinamycin	82 (94.3)	22 (88.0)	19 (95.0)	13 (100.0)	7 (87.5)	7 (100.0)	2 (100.0)
	Gentamicin	224 (30.7)	2 (1.3)	136 (91.9)	22 (15.5)	1 (1.1)	45 (95.7)	1 (2.1)
	Tobramycin	333 (45.6)	2 (1.3)	143 (96.6) 114 (79.7)	1 (1.1)	46 (95.8)	0 (0.0)
	Amikacin	21 (2.9)	1 (0.7)	2 (1.4)	0 (0.0)	1 (1.1)	15 (30.6)	0 (0.0)
	Minocycline	69 (9.4)	2 (1.3)	19 (12.8)	10 (6.9)	18 (18.9)	2 (4.1)	6 (12.5)
	Doxycycline	176 (42.8)	4 (4.8)	63 (63.6)	54 (67.5)	10 (21.7)	14 (48.3)	14 (66.7)
	Tigecycline	202 (27.9)	11 (7.4)	66 (45.2)	66 (46.5)	15 (15.8)	23 (47.9)	2 (4.3)
	Erythromycin	463 (64.1)	88 (58.7)	143 (97.3)	6 (4.3)	90 (95.7)	44 (91.7)	41 (85.4)
	Linezolid	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
	Vancomycin	465 (63.8)	91 (60.7)	46 (31.1)	108 (76.1)	81 (85.3)	43 (87.8)	45 (93.8)

*

Acknowledgments:

The authors would like to thank Koucila Chekhrit and Clémence Bruyere for their help in data collection and Dr Karen Pickett for her editorial suggestions.

Maps were prepared with Articque, Make your Map, freely available on the internet and d-maps.com.

The authors would like to thank M.C. Gaps and J.M. Ballec for their conscientious scientific assistance.

Conflict of interest: None

Funding: David Lebeaux was supported by the following grant: Bourse Junior 2015-Société de Pathologie Infec euse de angue Fran aise (SPILF).