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The file contains supplementary results showing that ICSN3250 specifically inhibited mTORC1 pathway.

The mTOR is a central regulator of cell growth and is highly activated in cancer cells to allow rapid tumor growth. The use of mTOR inhibitors as anticancer therapy has been approved for some types of tumors, albeit with modest results. We recently reported the synthesis of ICSN3250, a halitulin analogue with enhanced cytotoxicity. We report here that ICSN3250 is a specific mTOR inhibitor that operates through a mechanism distinct from those described for previous mTOR inhibitors. ICSN3250 competed with and displaced phosphatidic acid from the FRB domain in mTOR, thus preventing mTOR activation and leading to cytotoxicity. Docking and molecular dynamics simulations evidenced not only the high conformational plasticity of the FRB domain, but also the specific interactions of both ICSN3250 and phosphatidic acid with the FRB domain in mTOR. Furthermore, ICSN3250 toxicity was shown to act specifically in cancer cells, as noncancer cells showed up to 100-fold less sensitivity to ICSN3250, in contrast to other mTOR inhibitors that did not show selectivity. Thus, our results define ICSN3250 as a new class of mTOR inhibitors that specifically targets cancer cells.Significance: ICSN3250 defines a new class of mTORC1 inhibitors that displaces phosphatidic acid at the FRB domain of mTOR, inducing cell death specifically in cancer cells but not in noncancer cells. Cancer Res; 78(18); 5384–97. ©2018 AACR.

The file contains supplementary material and methods, as well as the legends for Supplementary Figures 1-7.

International audience; OXA-48-producing Enterobacterales have now widely disseminated throughout the world. Several variants have now been reported, differing by just a few amino-acid substitutions or deletions, mostly in the region of the loop β5-β6. As OXA-48 hydrolyzes carbapenems but lacks significant expanded-spectrum cephalosporin (ESC) hydrolytic activity, ESCs were suggested as a therapeutic option. Here, we have characterized OXA-517, a natural variant of OXA-48- with an Arg214Lys substitution and a deletion of Ile215 and Glu216 in the β5-β6 loop, capable of hydrolyzing at the same time ESC and carbapenems. MICs values of E. coli expressing blaOXA-517 gene revealed reduced susceptibility to carbapenems (similarly to OXA-48) and resistance to ESCs. Steady-state kinetic parameters revealed high catalytic efficiencies for ESCs and carbapenems. The blaOXA-517 gene was located on a ca. 31-kb plasmid identical to the prototypical IncL blaOXA-48-carrying plasmid except for an IS1R-mediated deletion of 30.7-kb in the tra operon. The crystal structure of OXA-517, determined to 1.86 Å resolution, revealed an expanded active site compared to that of OXA-48, which allows for accommodation of the bulky ceftazidime substrate. Our work illustrates the remarkable propensity of OXA-48-like carbapenemases to evolve through mutation/deletion in the β5-β6 loop to extend its hydrolysis profile to encompass most β-lactam substrates.

Gram-negative bacteria express structurally diverse lipoproteins in their cell envelope. Here, we find that approximately half of lipoproteins destined to the Escherichia coli outer membrane display an intrinsically disordered linker at their N terminus. Intrinsically disordered regions are common in proteins, but establishing their importance in vivo has remained challenging. As we sought to unravel how lipoproteins mature, we discovered that unstructured linkers are required for optimal trafficking by the Lol lipoprotein sorting system, whereby linker deletion re-routes three unrelated lipoproteins to the inner membrane. Focusing on the stress sensor RcsF, we found that replacing the linker with an artificial peptide restored normal outer-membrane targeting only when the peptide was of similar length and disordered. Overall, this study reveals the role played by intrinsic disorder in lipoprotein sorting, providing mechanistic insight into the biogenesis of these proteins and suggesting that evolution can select for intrinsic disorder that supports protein function. Approximately half of lipoproteins destined to the Escherichia coli outer membrane display intrinsically disordered linker peptides at their N termini, which are required for optimal trafficking by the Lol lipoprotein sorting system.

Class C β-lactamases or cephalosporinases can be classified into two functional groups (1, 1e) with considerable molecular variability (≤20% sequence identity). These enzymes are mostly encoded by chromosomal and inducible genes and are widespread among bacteria, including Proteobacteria in particular. Molecular identification is based principally on three catalytic motifs ((64)SXSK, (150)YXN, (315)KTG), but more than 70 conserved amino-acid residues (≥90%) have been identified, many close to these catalytic motifs. Nevertheless, the identification of a tiny, phylogenetically distant cluster (including enzymes from the genera Legionella, Bradyrhizobium, and Parachlamydia) has raised questions about the possible existence of a C2 subclass of β-lactamases, previously identified as serine hydrolases. In a context of the clinical emergence of extended-spectrum AmpC β-lactamases (ESACs), the genetic modifications observed in vivo and in vitro (point mutations, insertions, or deletions) during the evolution of these enzymes have mostly involved the Ω- and H-10/R2-loops, which vary considerably between genera, and, in some cases, the conserved triplet (150)YXN. Furthermore, the conserved deletion of several amino-acid residues in opportunistic pathogenic species of Acinetobacter, such as A. baumannii, A. calcoaceticus, A. pittii and A. nosocomialis (deletion of residues 304–306), and in Hafnia alvei and H. paralvei (deletion of residues 289–290), provides support for the notion of natural ESACs. The emergence of higher levels of resistance to β-lactams, including carbapenems, and to inhibitors such as avibactam is a reality, as the enzymes responsible are subject to complex regulation encompassing several other genes (ampR, ampD, ampG, etc.). Combinations of resistance mechanisms may therefore be at work, including overproduction or change in permeability, with the loss of porins and/or activation of efflux systems.

The EPIC consortium brings together experts from a wide range of fields that include clinical, molecular and basic microbiology, infectious diseases, computational biology and chemistry, drug discovery and design, bioinformatics, biochemistry, biophysics, pharmacology, toxicology, veterinary sciences, environmental sciences, and epidemiology. The main question to be answered by the EPIC alliance is the following: “What is the best approach for data mining on carbapenemase inhibitors and how to translate this data into experiments?” From this forum, we propose that the scientific community think up new strategies to be followed for the discovery of new carbapenemase inhibitors, so that this process is efficient and capable of providing results in the shortest possible time and within acceptable time and economic costs.

SUMMARYHsp60 chaperonins and their Hsp10 cofactors assist protein folding in all living cells, constituting the paradigmatic example of molecular chaperones. Despite extensive investigations of their structure and mechanism, crucial questions regarding how these chaperonins promote folding remain unsolved. Here, we report that the bacterial Hsp60 chaperonin GroEL forms a stable, functionally relevant complex with the chaperedoxin CnoX, a protein combining a chaperone and a redox function. Binding of GroES (Hsp10) to GroEL induces CnoX release. Cryo-electron microscopy provided crucial structural information on the GroEL-CnoX complex, showing that CnoX binds GroEL outside the substrate-binding site via a highly conserved C-terminal α-helix. Furthermore, the identification of complexes in which CnoX, bound to GroEL, forms mixed-disulfides with GroEL substrates indicates that CnoX likely functions as a redox quality-control plugin for GroEL. Proteins sharing structural features with CnoX exist in eukaryotes, which suggests that Hsp60 molecular plugins have been conserved through evolution.

International audience; OXA-48 carbapenemase has rapidly spread in many countries worldwide with several OXA-48-variants being described, differing by a few amino acid (AA) substitutions or deletions, mostly in the β5-β6 loop. While single AA substitutions have only a minor impact on OXA-48 hydrolytic profiles, others with 4 AA deletions result in loss of carbapenem hydrolysis and gain of expanded-spectrum cephalosporin (ESC) hydrolysis. We have replaced the β5-β6 loop of OXA-48 with that of OXA-18, a clavulanic-acid inhibited oxacillinase capable of hydrolyzing ESCs but not carbapenems. The hybrid enzyme OXA-48Loop18 was able to hydrolyze ESCs and carbapenems (although with a lower kcat), even though the β5-β6 loop was longer and its sequence quite different from that of OXA-48. The kinetic parameters of OXA-48Loop18 were in agreement with the MIC values. X-ray crystallography and molecular modeling suggest that the conformation of the grafted loop allows the binding of bulkier substrates, unlike that of the native loop, expanding the hydrolytic profile. This seems to be due not only to differences in AA sequence, but also to the backbone conformation the loop can adopt. Finally, our results provide further experimental evidence for the role of the β5-β6 loop in substrate selectivity of OXA-48-like enzymes and additional details on the structure-function relationship of β-lactamases, demonstrating how localized changes in these proteins can alter or expand their function, highlighting their plasticity.

Dans les années 1980, la prescription des premières céphalosporines de troisième génération (C3G) en médecine humaine a provoqué la sélection de mutants ou variants de pénicillinases de type TEM ou SHV dénommées : « Bêta-Lactamases à Spectre Elargi/Etendu » ou BLSE. Dans les années 1990 ont émergé de nouvelles enzymes ou CéfoTaXimases-München (CTX-M), transférables, inactivant les C3G, certaines étant devenues pandémiques. Elles sont maintenant retrouvées dans les trois écosystèmes (homme, animal, environnement) et leur grande diffusivité est liée à l’insertion du gène blaCTX-M (plasmide, transposon, intégron, séquence d’insertion) et à son association à d’autres gènes : des gènes de résistance par exemple aux fluoroquinolones et/ou aminoglycosides, mais aussi des gènes de virulence. Ces phénomènes ont conduit à la notion de clone majoritaire, en particulier chez l’homme, comme Escherichia coli ST131 du groupe phylogénétique B2 et du sérotype O25b ou H4 producteur du variant CTX-M-15. Ces gènes, d’une grande diversité, retrouvés chez de nombreux animaux (volailles, bovins, ovins…, voire les coquillages et les mouches) sont issus d’entérobactéries du genre Kluyvera dont la taxonomie et l’habitat restent à être mieux précisés. Mots clés : bêta-lactamase, BLSE, CTX-M, diversité, phylogénie, diffusion, homme, animal, environnement

Significance Hydrogen peroxide plays an important role in the fine balance between physiological and pathological processes. To detect this diffusible small molecule, we expanded the scope of organic triggers in developing borinic acids as an alternative and more sensitive trigger than the most conventional boronate-based sensors. We discovered that borinic acid is 10,000-fold more reactive than its boronic counterpart toward H 2 O 2 -mediated oxidation. An accurate determination of oxidation kinetic constants and computational experiments corroborate this higher reactivity. This improvement also proved effective for in-cell detection of exogenously as well as endogenously produced H 2 O 2 . We believe borinic acids represent a new and efficient tool allowing for the development of new devices for a better understanding of H 2 O 2 -mediated signaling processes.

Objectives Detection of carbapenemase-producing Enterobacterales (CPEs) is sometimes difficult with AmpC-hyperproducing Enterobacterales (AHEs), as they may falsely be classified as CPEs. Here, we present a rapid Carbapenem Inactivation Method (rCIM) optimized for AmpC producers (rCIM-A) that allows rapid and easy discrimination between AHEs and CPEs. Methods Enterobacterales (n = 249), including natural AmpC producers, AHEs, CPEs and non-carbapenemase-producing carbapenem-resistant control strains were evaluated, using Carba NP, rCIM and rCIM-A. The rCIM-A differs from the rCIM by the addition of cloxacillin (400 μg/mL) to the initial antibiotic incubation step. Results The rCIM-A yielded a sensitivity and specificity of 84.26% (95% CI: 76.00%–90.55%) and 99.29% (95% CI: 96.11%–99.98%), respectively, while those of the rCIM were 86.11% (95% CI: 78.13%–92.01%) and 80.85% (95% CI: 73.38%–86.99%), respectively; those of Carba NP were lower at 84.04% (95% CI: 75.05%–90.78%) and 91.37% (95% CI: 85.41%–95.46%), respectively, due to indeterminate results. The rCIM-A was capable of discriminating between AHEs and true CPEs, but still failed to identify OXA-23-producing Proteus mirabilis isolates and remained only partially reliable for identifying IMI-like producers and a few MBL (2 NDM-1, 1 LMB-1, 1 TMB-1 and 1 IMP-13) producers. One chromosomally encoded AmpC variant, MIR-10, gave repeatedly positive results using all three tests and was thus considered a false positive. Conclusions Specificity for AHEs greatly improved with the rCIM-A without altering the test performance for the other resistance mechanisms. It may replace the rCIM as a cheap, easy, rapid and accurate CPE detection test.

International audience; Over the last two decades, antimicrobial resistance has become a global health problem. In Gram-negative bacteria, metallo-β-lactamases (MBLs), which inactivate virtually all β-lactams, increasingly contribute to this phenomenon. The aim of this study is to characterize VIM-52, a His224Arg variant of VIM-1, identified in a Klebsiella pneumoniae clinical isolate. VIM-52 conferred lower MICs to cefepime and ceftazidime as compared to VIM-1. These results were confirmed by steady state kinetic measurements, where VIM-52 yielded a lower activity towards ceftazidime and cefepime but not against carbapenems. Residue 224 is part of the L10 loop (residues 221-241), which borders the active site. As Arg 224 and Ser 228 are both playing an important and interrelated role in enzymatic activity, stability and substrate specificity for the MBLs, targeted mutagenesis at both positions were performed and further confirmed their crucial role for substrate specificity.

International audience; The occurrence of resistances in Gram negative bacteria is steadily increasing to reach extremely worrying levels and one of the main causes of resistance is the massive spread of very efficient β-lactamases which render most β-lactam antibiotics useless. Herein, we report the development of a series of imino-analogues of β-lactams (namely azetidinimines) as efficient non-covalent inhibitors of β-lactamases. Despite the structural and mechanistic differences between serine-β-lactamases KPC-2 and OXA-48 and metallo-β-lactamase NDM-1, all three enzymes can be inhibited at a submicromolar level by compound 7dfm, which can also repotentiate imipenem against a resistant strain of Escherichia coli expressing NDM-1. We show that 7dfm can efficiently inhibit not only the three main clinically-relevant carbapenemases of Ambler classes A (KPC-2), B (NDM-1) and D (OXA-48) with Ki’s below 0.3 μM, but also the cephalosporinase CMY-2 (class C, 86% inhibition at 10 μM). Our results pave the way for the development of a new structurally original family of non-covalent broad-spectrum inhibitors of β-lactamases.

BackgroundKPC-like carbapenemases have spread worldwide with more than 30 variants identified that differ by single or double amino-acid substitutions.ObjectivesTo describe the steady-state kinetic parameters of KPC-28, which differs from KPC-2 by a H274Y substitution and the deletion of two amino acids (Δ242-GT-243).MethodsThe blaKPC-2, blaKPC-3, blaKPC-14 and blaKPC-28 genes were cloned into a pTOPO vector for susceptibility testing or into pET41b for overexpression, purification and subsequent kinetic parameter (Km, kcat) determination. Molecular docking experiments were performed to explore the role of the amino-acid changes in the carbapenemase activity.ResultsSusceptibility testing revealed that Escherichia coli producing KPC-28 displayed MICs that were lower for carbapenems and higher for ceftazidime and ceftazidime/avibactam as compared with KPC-2. The catalytic efficiencies of KPC-28 and KPC-14 for imipenem were 700-fold and 200-fold lower, respectively, than those of KPC-2, suggesting that Δ242-GT-243 in KPC-28 and KPC-14 is responsible for reduced carbapenem hydrolysis. Similarly, the H274Y substitution resulted in KPC-28 in a 50-fold increase in ceftazidime hydrolysis that was strongly reversed by clavulanate.ConclusionsWe have shown that KPC-28 lacks carbapenemase activity, has increased ceftazidime hydrolytic activity and is strongly inhibited by clavulanate. KPC-28-producing E. coli isolates display an avibactam-resistant ESBL profile, which may be wrongly identified by molecular and immunochromatographic assays as the presence of a carbapenemase. Accordingly, confirmation of carbapenem hydrolysis will be mandatory with assays based solely on blaKPC gene or gene product detection.

International audience; Colletotrichum higginsianum is the causal agent of crucifer anthracnose disease, responsible for important economic losses in Brassica crops. A mutant lacking the CclA subunit of the COMPASS complex was expected to undergo chromatin decondensation and the activation of cryptic secondary metabolite biosynthetic gene clusters. Liquid-state fermentation of the Δ cclA mutant coupled with in situ solid-phase extraction led to the production of three families of compounds, namely, colletorin and colletochlorin derivatives with two new representatives, colletorin D (1) and colletorin D acid (2), the diterpenoid α-pyrone higginsianin family with two new analogues, higginsianin C (3) and 13- epi-higginsianin C (4), and sclerosporide (5) coupling a sclerosporin moiety with dimethoxy inositol.

Mavacamten (MYK-461) is a small-molecule allosteric inhibitor of sarcomeric myosins being used in preclinical/clinical trials for hypertrophic cardiomyopathy treatment. A better understanding of its impact on force generation in intact or skinned striated muscle preparations, especially for human cardiac muscle, has been hindered by diffusional barriers. These limitations have been overcome by mechanical experiments using myofibrils subject to perturbations of the contractile environment by sudden solution changes. Here, we characterize the action of mavacamten in human ventricular myofibrils compared with fast skeletal myofibrils from rabbit psoas. Mavacamten had a fast, fully reversible, and dose-dependent negative effect on maximal Ca2+-activated isometric force at 15°C, which can be explained by a sudden decrease in the number of heads functionally available for interaction with actin. It also decreased the kinetics of force development in fast skeletal myofibrils, while it had no effect in human ventricular myofibrils. For both myofibril types, the effects of mavacamten were independent from phosphate in the low-concentration range. Mavacamten did not alter force relaxation of fast skeletal myofibrils, but it significantly accelerated the relaxation of human ventricular myofibrils. Lastly, mavacamten had no effect on resting tension but inhibited the ADP-stimulated force in the absence of Ca2+. Altogether, these effects outline a motor isoform-specific dependence of the inhibitory effect of mavacamten on force generation, which is mediated by a reduction in the availability of strongly actin-binding heads. Mavacamten may thus alter the interplay between thick and thin filament regulation mechanisms of contraction in association with the widely documented drug effect of stabilizing myosin motor heads into autoinhibited states.

BackgroundOXA-48-producing Enterobacterales have widely disseminated globally with an increasing number of variants identified. Among them, OXA-244 is increasingly reported, despite detection difficulties.ObjectivesTo determine the steady-state kinetic parameters of OXA-244.MethodsThe blaOXA-244 gene was amplified, cloned into plasmids p-TOPO and pET41b+, and transformed into Escherichia coli TOP10 for MIC determination and E. coli BL21 DE3 for purification. Steady-state kinetic parameters and IC50s of clavulanic acid, tazobactam and NaCl were determined using purified OXA-244. Molecular modelling was also performed.ResultsA reduction in MICs of temocillin and carbapenems was observed in E. coli expressing OXA-244 as compared with OXA-48. The kinetic parameters revealed a reduced carbapenemase activity of OXA-244 as compared with OXA-48, especially for imipenem, which was 10-fold lower. Similarly, catalytic efficiency (kcat/Km) was reduced by 4-fold and 20-fold for ampicillin and temocillin, respectively. Kinetic parameters for cephalosporins were, however, similar. Molecular modelling studies evidenced the key role of R214 in OXA-48, establishing salt bridges with D159 and with the carboxylate group of the R1 substituent of temocillin. These interactions are not possible with G214 in OXA-244, explaining the reduced affinity of temocillin for this enzyme. The R214G mutation in OXA-244 is also likely to induce changes in the active site’s water network that would explain the decrease in the hydrolysis rate of carbapenems.ConclusionsOur data confirm that the R214G mutation (present in OXA-244) results in reduced carbapenem- and temocillin-hydrolysing activity, confirming the crucial role of residue 214 in the hydrolysis of these substrates by OXA-48-like β-lactamases.

In this study, Kawai et al. address the role of inorganic phosphate in muscle cross-bridge kinetics using a subcellular structure called myofibrils. They demonstrate that phosphate binding is a key determinant of oscillatory work and suggest that it may also compete with ATP for binding to the myosin head., In this study, we aimed to study the role of inorganic phosphate (Pi) in the production of oscillatory work and cross-bridge (CB) kinetics of striated muscle. We applied small-amplitude sinusoidal length oscillations to rabbit psoas single myofibrils and muscle fibers, and the resulting force responses were analyzed during maximal Ca2+ activation (pCa 4.65) at 15°C. Three exponential processes, A, B, and C, were identified from the tension transients, which were studied as functions of Pi concentration ([Pi]). In myofibrils, we found that process C, corresponding to phase 2 of step analysis during isometric contraction, is almost a perfect single exponential function compared with skinned fibers, which exhibit distributed rate constants, as described previously. The [Pi] dependence of the apparent rate constants 2πb and 2πc, and that of isometric tension, was studied to characterize the force generation and Pi release steps in the CB cycle, as well as the inhibitory effect of Pi. In contrast to skinned fibers, Pi does not accumulate in the core of myofibrils, allowing sinusoidal analysis to be performed nearly at [Pi] = 0. Process B disappeared as [Pi] approached 0 mM in myofibrils, indicating the significance of the role of Pi rebinding to CBs in the production of oscillatory work (process B). Our results also suggest that Pi competitively inhibits ATP binding to CBs, with an inhibitory dissociation constant of ∼2.6 mM. Finally, we found that the sinusoidal waveform of tension is mostly distorted by second harmonics and that this distortion is closely correlated with production of oscillatory work, indicating that the mechanism of generating force is intrinsically nonlinear. A nonlinear force generation mechanism suggests that the length-dependent intrinsic rate constant is asymmetric upon stretch and release and that there may be a ratchet mechanism involved in the CB cycle.

International audience; Significant inhibition of Aurora B was achieved by the synthesis of simplified fragments of benzosceptrins and oroidin belonging to the marine pyrrole-2-aminoimidazoles metabolites isolated from sponges. Evaluation of kinase inhibition enabled the discovery of a synthetically accessible rigid acetylenic structural analogue EL-228 (1), whose structure could be optimized into the potent CJ2-150 (37). Here we present the synthesis of new inhibitors of Aurora B kinase, which is an important target for cancer therapy through mitosis regulation. The biologically oriented synthesis yielded several nanomolar inhibitors. The optimized compound CJ2-150 (37) showed a non-ATP competitive allosteric mode of action in a mixed-type inhibition for Aurora B kinase. Molecular docking identified a probable binding mode in the allosteric site "F" and highlighted the key interactions with the protein. We describe the improvement of the inhibitory potency and specificity of the novel scaffold as well as the characterization of the mechanism of action.; Une inhibition significative d'Aurora B a été obtenue par la synthèse de fragments simplifiés de benzosceptrines et d'oroïdine appartenant aux métabolites marins pyrrole-2-aminoimidazoles isolés à partir d'éponges. L'évaluation de l'inhibition de la kinase a permis la découverte d'un analogue structurel acétylénique rigide synthétiquement accessible EL-228 (1), dont la structure pourrait être optimisée dans le puissant CJ2-150 (37). Nous présentons ici la synthèse de nouveaux inhibiteurs de l'Aurora B kinase, qui est une cible importante pour le traitement du cancer par la régulation de la mitose. La synthèse biologiquement orientée a donné plusieurs inhibiteurs nanomolaires. Le composé optimisé CJ2-150 (37) a montré un mode d'action allostérique compétitif non ATP dans une inhibition de type mixte de la kinase Aurora B. L'arrimage moléculaire a identifié un mode de liaison probable dans le site allostérique «F» et a mis en évidence les interactions clés avec la protéine. Nous décrivons l'amélioration de la puissance inhibitrice et la spécificité du nouvel échafaudage ainsi que la caractérisation du mécanisme d'action.

We thank Asma Boujtat for technical help. We are indebted to the members of the Collet laboratory and to Nassos Typas (EMBL, Heidelberg) for helpful suggestions and discussions and to Tom Silhavy (Princeton) for providing bacterial strains. J.S. was a research fellow of the FRIA and J.F.C. is an Investigator of the FRFS-WELBIO. This work was funded by the WELBIO, by grants from the F.R.S.-FNRS, from the Fédération Wallonie-Bruxelles (ARC 17/22-087), from the European Commission via the International Training Network Train2Target (721484), and from the EOS Excellence in Research Program of the FWO and FRS-FNRS (G0G0818N).; Gram-negative bacteria express structurally diverse lipoproteins in their envelope. Here we found that approximately half of lipoproteins destined to the Escherichia coli outer membrane display an intrinsically disordered linker at their N-terminus. Intrinsically disordered regions are common in proteins, but establishing their importance in vivo has remained challenging. Here, as we sought to unravel how lipoproteins mature, we discovered that unstructured linkers are required for optimal trafficking by the Lol lipoprotein sorting system: linker deletion re-routes three unrelated lipoproteins to the inner membrane. Focusing on the stress sensor RcsF, we found that replacing the linker with an artificial peptide restored normal outer membrane targeting only when the peptide was of similar length and disordered. Overall, this study reveals the role played by intrinsic disorder in lipoprotein sorting, providing mechanistic insight into the biogenesis of these proteins and suggesting that evolution can select for intrinsic disorder that supports protein function.

Resistance to the ceftazidime (CAZ)-avibactam (AVI) combination is increasingly being reported. Here, we report a CAZ-AVI-resistant Klebsiella pneumoniae strain belonging to the high-risk sequence type 307 (ST307) clone and producing Klebsiella pneumoniae carbapenemase 39 (KPC-39), a single-amino-acid variant of KPC-3 (A172T). Cloning experiments, steady-state kinetic parameters, and molecular dynamics simulations revealed a loss of carbapenemase activity and increased affinity for CAZ. KPC-39 was identified in a patient without prior exposure to CAZ-AVI, suggesting silent dissemination in European health care settings.

International audience; We investigated the presence of carbapenemases in carbapenem-resistant Pseudomonas aeruginosa isolates, which were collected over a 14-month period in a Turkish hospital, with in-depth molecular characterization of carbapenemase-producing isolates. Among 45 study isolates, 2 isolates were identified as carbapenemase producers by both Carba NP and Carbapenem Inactivation Method tests, and only 1 of them gave a positive result in polymerase chain reaction tests for a carbapenemase gene (blaVIM). Whole genome sequencing of the 2 isolates revealed the presence of blaVIM-5 gene in an ST308 isolate, while the other one expressed IMP-7 in an ST357 isolate; both STs are considered high-risk clones. The 2 carbapenemase-producing isolates were multidrug resistant, as they harbored other resistance determinants, including a variant of the recently described plasmid-encoded fluoroquinolone resistance determinant crpP gene, crpP-2. We report for the first time P. aeruginosa high-risk clones carrying VIM-5– and IMP-7–type carbapenemases with multiple resistance determinants in Turkey.