Identification of Diverse Integron and Plasmid Structures Carrying a Novel Carbapenemase Among Pseudomonas Species

A novel carbapenem-hydrolyzing beta-lactamase, called IMP-63, was identified in three clonally distinct strains of Pseudomonas aeruginosa and two strains of Pseudomonas putida isolated within a 4 year timeframe in three French hospitals. The blaIMP–63 gene that encodes this carbapenemase turned out to be located in the variable region of four integrons (In1297, In1574, In1573, and In1572) and to coexist with novel or rare gene cassettes (fosM, gcu170, gcuF1) and insertion elements (ISPsp7v, ISPa16v). All these integrons except one (In1574) were flanked by a copy of insertion sequence ISPa17 next to the orf6 putative gene, and were carried by non-conjugative plasmids (pNECK1, pROUSS1, pROUSS2, pROUE1). These plasmids exhibit unique modular structures and partial sequence homologies with plasmids previously identified in various non-fermenting environmental Gram-negative species. Lines of evidence suggest that ISPa17 promoted en bloc the transposition of IMP-63-encoding integrons on these different plasmids. As demonstrated by genotyping experiments, isolates of P. aeruginosa harboring the 28.9-kb plasmid pNECK1 and belonging to international “high-risk” clone ST308 were responsible for an outbreak in one hospital. Collectively, these data provide an insight into the complex and unpredictable routes of diffusion of some resistance determinants, here blaIMP–63, among Pseudomonas species

p27 controls autophagic vesicle trafficking in glucose-deprived cells via the regulation of ATAT1-mediated microtubule acetylation.

peer reviewedThe cyclin-dependent kinase inhibitor p27Kip1 (p27) has been involved in promoting autophagy and survival in conditions of metabolic stress. While the signaling cascade upstream of p27 leading to its cytoplasmic localization and autophagy induction has been extensively studied, how p27 stimulates the autophagic process remains unclear. Here, we investigated the mechanism by which p27 promotes autophagy upon glucose deprivation. Mouse embryo fibroblasts (MEFs) lacking p27 exhibit a decreased autophagy flux compared to wild-type cells and this is correlated with an abnormal distribution of autophagosomes. Indeed, while autophagosomes are mainly located in the perinuclear area in wild-type cells, they are distributed throughout the cytoplasm in p27-null MEFs. Autophagosome trafficking towards the perinuclear area, where most lysosomes reside, is critical for autophagosome-lysosome fusion and cargo degradation. Vesicle trafficking is mediated by motor proteins, themselves recruited preferentially to acetylated microtubules, and autophagy flux is directly correlated to microtubule acetylation levels. p27-/- MEFs exhibit a marked reduction in microtubule acetylation levels and restoring microtubule acetylation in these cells, either by re-expressing p27 or with deacetylase inhibitors, restores perinuclear positioning of autophagosomes and autophagy flux. Finally, we find that p27 promotes microtubule acetylation by binding to and stabilizing α-tubulin acetyltransferase (ATAT1) in glucose-deprived cells. ATAT1 knockdown results in random distribution of autophagosomes in p27+/+ MEFs and impaired autophagy flux, similar to that observed in p27-/- cells. Overall, in response to glucose starvation, p27 promotes autophagy by facilitating autophagosome trafficking along microtubule tracks by maintaining elevated microtubule acetylation via an ATAT1-dependent mechanism

Loss of p27/Kip1 promotes metaplasia in the pancreas via the regulation of Sox9 expression.

p27Kip1 (p27) is a negative regulator of proliferation and a tumor suppressor via the inhibition of cyclin-CDK activity in the nucleus. p27 is also involved in the regulation of other cellular processes, including transcription by acting as a transcriptional co-repressor. Loss of p27 expression is frequently observed in pancreatic adenocarcinomas in human and is associated with decreased patient survival. Similarly, in a mouse model of K-Ras-driven pancreatic cancer, loss of p27 accelerates tumor development and shortens survival, suggesting an important role for p27 in pancreatic tumorigenesis. Here, we sought to determine how p27 might contribute to early events leading to tumor development in the pancreas. We found that K-Ras activation in the pancreas causes p27 mislocalization at pre-neoplastic stages. Moreover, loss of p27 or expression of a mutant p27 that does not bind cyclin-CDKs causes the mislocalization of several acinar polarity markers associated with metaplasia and induces the nuclear expression of Sox9 and Pdx1 two transcription factors involved in acinar-to-ductal metaplasia. Finally, we found that p27 directly represses transcription of Sox9, but not that of Pdx1. Thus, our results suggest that K-Ras activation, the earliest known event in pancreatic carcinogenesis, may cause loss of nuclear p27 expression which results in derepression of Sox9, triggering reprogramming of acinar cells and metaplasia

Genomic characterization of an NDM-9-producing Acinetobacter baumannii clinical isolate and role of Glu152Lys substitution in the enhanced cefiderocol hydrolysis of NDM-9

Here, we characterized the first French NDM-9-producing Acinetobacter baumannii isolate. A. baumannii 13A297, which belonged to the STPas25 (international clone IC7), was highly resistant to β-lactams including cefiderocol (MIC >32 mg/L). Whole genome sequencing (WGS) using both Illumina and Oxford Nanopore technologies revealed a 166-kb non-conjugative plasmid harboring a blaNDM-9 gene embedded in a Tn125 composite transposon. Complementation of E. coli DH5α and A. baumannii CIP70.10 strains with the pABEC plasmid carrying the blaNDM-1 or blaNDM-9 gene, respectively, resulted in a significant increase in cefiderocol MIC values (16 to >256-fold), particularly in the NDM-9 transformants. Interestingly, steady-state kinetic parameters, measured using purified NDM-1 and NDM-9 (Glu152Lys) enzymes, revealed that the affinity for cefiderocol was 3-fold higher for NDM-9 (Km = 53 μM) than for NDM-1 (Km = 161 μM), leading to a 2-fold increase in catalytic efficiency for NDM-9 (0.13 and 0.069 μM−1.s−1, for NDM-9 and NDM-1, respectively). Finally, we showed by molecular docking experiments that the residue 152 of NDM-like enzymes plays a key role in cefiderocol binding and resistance, by allowing a strong ionic interaction between the Lys152 residue of NDM-9 with both the Asp223 residue of NDM-9 and the carboxylate group of the R1 substituent of cefiderocol

Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Investigation of new functions of p27 in oncogenesis and cell invasion

Le cycle cellulaire est un processus finement régulé à différents niveaux. p27 est un inhibiteur des complexes cyclines/CDK et cette fonction lui confère un rôle antiprolifératif lorsqu'il est localisé dans le noyau. De nombreuses études ont montré que p27 se comporte comme un suppresseur de tumeur lorsqu'il est localisé dans le noyau des cellules via l'inhibition des cyclines/CDK. De manière surprenante, p27 a également des fonctions oncogéniques dans certaines situations, notamment lorsqu'il est présent dans le cytoplasme. Il s'avère qu'en plus de ses fonctions de régulateur du cycle cellulaire, p27 est également impliqué dans la régulation d'autres processus cellulaires, comme la migration, la cytocinèse, la transcription ou encore l'autophagie et la différenciation. Durant ma thèse, j'ai caractérisé deux de ces nouvelles fonctions en mettant en lumière un rôle de p27 dans la régula.on des invadopodes ainsi que dans l'oncogenèse pancréatique.Mon premier objectif a été de comprendre la fonction de l'interaction de p27 avec la protéine Cortactine, un nouveau partenaire de p27.Cette protéine joue un rôle important dans la régulation de l'invasion cellulaire. J'ai confirmé cette interaction dans différents types cellulaires et observé une colocalisation au niveau des invadopodes. J'ai également cartographié leurs domaines d'interaction respectifs et montré que l'interaction de p27 avec Cortactine était induite par une stimulation avec du sérum. L'interaction p27/Cortactine permet le recrutement de PAK1, une sérine/thréonine kinase impliqué dans l'invasion cellulaire et favorisant notamment le renouvellement des invadopodes, sur Cortactine. J'ai également observé que les cellules invalidées pour p27 présentent de nombreux invadopodes et dégradent la matrice extracellulaire de manière très importante par rapport aux cellules exprimant p27. Par des approches d'inhibiteurs et de siARN j'ai mis en évidence que ce phénotype implique la voie Rac1/PAK1/phospho-Ser113-Cortactine qui est sous-activée en l'absence de p27 à cause du défaut de recrutement de PAK1 sur Cortactine, stabilisant ainsi les invadopodes. Mon second objectif a été d'étudier les mécanismes par lesquels p27 régule l'oncogenèse pancréatique. En effet, différentes études cliniques ou chez la souris ont montré que la localisation nucléaire de p27 était requise pour son rôle de suppresseur de tumeur dans le pancréas. Or nous avons constaté dans un modèle murin génétique d'oncogenèse pancréatique induite par l'activation de K-Ras que p27 était exclu du noyau avant l'apparition des premières lésions, suggérant un rôle précoce dans l'oncogenèse de ce tissu. De manière surprenante, l'étude comparative par immunomarquage de pancréas de souris p27+/+, p27-/- et p27CK-/CK-, un modèle de souris knock-in où p27 n'est plus capable d'inhiber les cyclines/CDK, n'a pas montré d'effet de p27 sur la prolifération dans le pancréas exocrine. Par contre, ces études ont montré une localisation anormale de plusieurs marqueurs de polarité acinaire ainsi qu'une réexpression des facteurs de transcription Sox9 et Pdx1, impliqués dans le processus de transdifférenciation cellulaire à l'origine de l'oncogenèse pancréatique appelée métaplasie acino-canalaire. Nous avons montré que p27 régulait de manière directe la transcription de Sox9 en interagissant avec son promoteur, suggérant que p27 participe normalement à la répression transcriptionnelle de Sox9 dans le noyau, réprimant ainsi la métaplasie acino-canalaire.Ainsi, mes travaux de thèse ont permis l'identification de deux nouvelles fonctions de p27. Une fonction oncogénique, indépendante de son activité d'inhibiteur des CDK/cyclines, par laquelle p27 régule la protéine Cortactine, et par ce biais les invadopodes et l'invasion ainsi qu'une fonction de suppresseur de tumeur dépendante des cyclines/CDK via la répression de la transcription de Sox9 dans les cellules acinaires du pancréas.Cell cycle is a tightly regulated process. One level of regulation is provided by p27, a cyclin/ CDK inhibitor and as such, p27 has an antiproliferative function. Several studies have shown that p27 is a tumor suppressor when it is located in the nucleus via the inhibition of cyclins/ CDKs. Surprisingly, p27 may also play oncogenic roles depending on the cellular context, especially when it is excluded from the nucleus. In fact, several lines of evidence indicate that p27 functions extend beyond cell cycle regulation and that p27 also regulates cell migration, cytokinesis, transcription, autophagy and stemness/differenciation. During my PhD, I have characterized two novel p27 functions, one in the regulation of invadopodia, and the second in pancreatic oncogenesis.My first aim was to investigate the function of the interaction between p27 and Cortactin, a new p27-interacting protein which is an important regulator of cell invasion. I confirmed this interaction in different cell lines and found that p27 and Cortactin colocalized in invadopodia. I have mapped the domains mediating the interaction in both proteins and found that this interaction is induced after serum stimulation. p27 allows the recruitment of PAK1, a serine/ threonine kinase involved in cell invasion which promotes invadopodia turnover, on Cortactin. I also found that p27 knock-out cells have an increased number of invadopodia and degrade extracellular matrix more efficiently than wild-type cells. Using inhibitors and siRNAs I have shown that this phenotype involves the Rac1/PAK1/phospho-Ser113-Cortac.n pathway, which is underactivated in absence of p27 due to the PAK1/Cortactin interaction defect, causing a stabilization of invadopodia.My second aim was to study the mechanism by which p27 regulates pancreatic oncogenesis. Severals studies in the clinic or in animal models have shown that nuclear localization of p27 is required for its tumor suppressor function in the pancreas. In a genetic mouse model of K- Ras driven pancreatic oncogenesis, I found that p27 was excluded from the nucleus before the apparition of lesions, suggesting an early function in oncogenesis in this tissue. Surprisingly, comparative studies of pancreas from p27+/+, p27-/- and p27CK-/CK- (a knock- in mouse model where p27 no longer binds cyclins/CDKs) mice by immunostaining did not show any difference in proliferation in the exocrine pancreas. However, these studies have shown the mislocalization of different acinar polarity markers and the re-expression of two transcription factors, Sox9 and Pdx1, which are involved in acinar-to-ductal metaplasia, a cell transdifferenciation process thought to be the underlying cause of pancreatic oncogenesis. I have found that p27 regulates Sox9 transcription and directly interacts with its promoter, suggesting that p27 participates in the transcriptionnal repression of Sox9 in normal conditions to prevent acinar-to-ductal metaplasia. Overall, my PhD work has allowed the identification of two novel roles of p27. An oncogenic function, independent of its cyclin/CDK inhibitory activity, by which p27 regulates Cortac.n, invadopodia and cell invasion and a tumor suppressor function dependent of cyclin/CDK via the repression of Sox9 transcription in pancreas acinar cells

Etude de nouvelles fonctions de p27 dans l'oncogenèse et l'invasion cellulaire

Le cycle cellulaire est un processus finement régulé à différents niveaux. p27 est un inhibiteur des complexes cyclines/CDK et cette fonction lui confère un rôle antiprolifératif lorsqu'il est localisé dans le noyau. De nombreuses études ont montré que p27 se comporte comme un suppresseur de tumeur lorsqu'il est localisé dans le noyau des cellules via l'inhibition des cyclines/CDK. De manière surprenante, p27 a également des fonctions oncogéniques dans certaines situations, notamment lorsqu'il est présent dans le cytoplasme. Il s'avère qu'en plus de ses fonctions de régulateur du cycle cellulaire, p27 est également impliqué dans la régulation d'autres processus cellulaires, comme la migration, la cytocinèse, la transcription ou encore l'autophagie et la différenciation. Durant ma thèse, j'ai caractérisé deux de ces nouvelles fonctions en mettant en lumière un rôle de p27 dans la régula.on des invadopodes ainsi que dans l'oncogenèse pancréatique.Mon premier objectif a été de comprendre la fonction de l'interaction de p27 avec la protéine Cortactine, un nouveau partenaire de p27.Cette protéine joue un rôle important dans la régulation de l'invasion cellulaire. J'ai confirmé cette interaction dans différents types cellulaires et observé une colocalisation au niveau des invadopodes. J'ai également cartographié leurs domaines d'interaction respectifs et montré que l'interaction de p27 avec Cortactine était induite par une stimulation avec du sérum. L'interaction p27/Cortactine permet le recrutement de PAK1, une sérine/thréonine kinase impliqué dans l'invasion cellulaire et favorisant notamment le renouvellement des invadopodes, sur Cortactine. J'ai également observé que les cellules invalidées pour p27 présentent de nombreux invadopodes et dégradent la matrice extracellulaire de manière très importante par rapport aux cellules exprimant p27. Par des approches d'inhibiteurs et de siARN j'ai mis en évidence que ce phénotype implique la voie Rac1/PAK1/phospho-Ser113-Cortactine qui est sous-activée en l'absence de p27 à cause du défaut de recrutement de PAK1 sur Cortactine, stabilisant ainsi les invadopodes. Mon second objectif a été d'étudier les mécanismes par lesquels p27 régule l'oncogenèse pancréatique. En effet, différentes études cliniques ou chez la souris ont montré que la localisation nucléaire de p27 était requise pour son rôle de suppresseur de tumeur dans le pancréas. Or nous avons constaté dans un modèle murin génétique d'oncogenèse pancréatique induite par l'activation de K-Ras que p27 était exclu du noyau avant l'apparition des premières lésions, suggérant un rôle précoce dans l'oncogenèse de ce tissu. De manière surprenante, l'étude comparative par immunomarquage de pancréas de souris p27+/+, p27-/- et p27CK-/CK-, un modèle de souris knock-in où p27 n'est plus capable d'inhiber les cyclines/CDK, n'a pas montré d'effet de p27 sur la prolifération dans le pancréas exocrine. Par contre, ces études ont montré une localisation anormale de plusieurs marqueurs de polarité acinaire ainsi qu'une réexpression des facteurs de transcription Sox9 et Pdx1, impliqués dans le processus de transdifférenciation cellulaire à l'origine de l'oncogenèse pancréatique appelée métaplasie acino-canalaire. Nous avons montré que p27 régulait de manière directe la transcription de Sox9 en interagissant avec son promoteur, suggérant que p27 participe normalement à la répression transcriptionnelle de Sox9 dans le noyau, réprimant ainsi la métaplasie acino-canalaire.Ainsi, mes travaux de thèse ont permis l'identification de deux nouvelles fonctions de p27. Une fonction oncogénique, indépendante de son activité d'inhibiteur des CDK/cyclines, par laquelle p27 régule la protéine Cortactine, et par ce biais les invadopodes et l'invasion ainsi qu'une fonction de suppresseur de tumeur dépendante des cyclines/CDK via la répression de la transcription de Sox9 dans les cellules acinaires du pancréas.Cell cycle is a tightly regulated process. One level of regulation is provided by p27, a cyclin/ CDK inhibitor and as such, p27 has an antiproliferative function. Several studies have shown that p27 is a tumor suppressor when it is located in the nucleus via the inhibition of cyclins/ CDKs. Surprisingly, p27 may also play oncogenic roles depending on the cellular context, especially when it is excluded from the nucleus. In fact, several lines of evidence indicate that p27 functions extend beyond cell cycle regulation and that p27 also regulates cell migration, cytokinesis, transcription, autophagy and stemness/differenciation. During my PhD, I have characterized two novel p27 functions, one in the regulation of invadopodia, and the second in pancreatic oncogenesis.My first aim was to investigate the function of the interaction between p27 and Cortactin, a new p27-interacting protein which is an important regulator of cell invasion. I confirmed this interaction in different cell lines and found that p27 and Cortactin colocalized in invadopodia. I have mapped the domains mediating the interaction in both proteins and found that this interaction is induced after serum stimulation. p27 allows the recruitment of PAK1, a serine/ threonine kinase involved in cell invasion which promotes invadopodia turnover, on Cortactin. I also found that p27 knock-out cells have an increased number of invadopodia and degrade extracellular matrix more efficiently than wild-type cells. Using inhibitors and siRNAs I have shown that this phenotype involves the Rac1/PAK1/phospho-Ser113-Cortac.n pathway, which is underactivated in absence of p27 due to the PAK1/Cortactin interaction defect, causing a stabilization of invadopodia.My second aim was to study the mechanism by which p27 regulates pancreatic oncogenesis. Severals studies in the clinic or in animal models have shown that nuclear localization of p27 is required for its tumor suppressor function in the pancreas. In a genetic mouse model of K- Ras driven pancreatic oncogenesis, I found that p27 was excluded from the nucleus before the apparition of lesions, suggesting an early function in oncogenesis in this tissue. Surprisingly, comparative studies of pancreas from p27+/+, p27-/- and p27CK-/CK- (a knock- in mouse model where p27 no longer binds cyclins/CDKs) mice by immunostaining did not show any difference in proliferation in the exocrine pancreas. However, these studies have shown the mislocalization of different acinar polarity markers and the re-expression of two transcription factors, Sox9 and Pdx1, which are involved in acinar-to-ductal metaplasia, a cell transdifferenciation process thought to be the underlying cause of pancreatic oncogenesis. I have found that p27 regulates Sox9 transcription and directly interacts with its promoter, suggesting that p27 participates in the transcriptionnal repression of Sox9 in normal conditions to prevent acinar-to-ductal metaplasia. Overall, my PhD work has allowed the identification of two novel roles of p27. An oncogenic function, independent of its cyclin/CDK inhibitory activity, by which p27 regulates Cortac.n, invadopodia and cell invasion and a tumor suppressor function dependent of cyclin/CDK via the repression of Sox9 transcription in pancreas acinar cells

Hypoxia-Induced Reactivity of Tumor-Associated Astrocytes Affects Glioma Cell Properties

Glioblastoma is characterized by extensive necrotic areas with surrounding hypoxia. The cancer cell response to hypoxia in these areas is well-described; it involves a metabolic shift and an increase in stem cell-like characteristics. Less is known about the hypoxic response of tumor-associated astrocytes, a major component of the glioma tumor microenvironment. Here, we used primary human astrocytes and a genetically engineered glioma mouse model to investigate the response of this stromal cell type to hypoxia. We found that astrocytes became reactive in response to intermediate and severe hypoxia, similarly to irradiated and temozolomide-treated astrocytes. Hypoxic astrocytes displayed a potent hypoxia response that appeared to be driven primarily by hypoxia-inducible factor 2-alpha (HIF-2α). This response involved the activation of classical HIF target genes and the increased production of hypoxia-associated cytokines such as TGF-β1, IL-3, angiogenin, VEGF-A, and IL-1 alpha. In vivo, astrocytes were present in proximity to perinecrotic areas surrounding HIF-2α expressing cells, suggesting that hypoxic astrocytes contribute to the glioma microenvironment. Extracellular matrix derived from hypoxic astrocytes increased the proliferation and drug efflux capability of glioma cells. Together, our findings suggest that hypoxic astrocytes are implicated in tumor growth and potentially stemness maintenance by remodeling the tumor microenvironment

Niche-derived soluble DLK1 promotes glioma growth

Tumor cell behaviors associated with aggressive tumor growth such as proliferation, therapeutic resistance, and stem cell characteristics are regulated in part by soluble factors derived from the tumor microenvironment. Tumor-associated astrocytes represent a major component of the glioma tumor microenvironment, and astrocytes have an active role in maintenance of normal neural stem cells in the stem cell niche, in part via secretion of soluble delta-like noncanonical Notch ligand 1 (DLK1). We found that astrocytes, when exposed to stresses of the tumor microenvironment such as hypoxia or ionizing radiation, increased secretion of soluble DLK1. Tumor-associated astrocytes in a glioma mouse model expressed DLK1 in perinecrotic and perivascular tumor areas. Glioma cells exposed to recombinant DLK1 displayed increased proliferation, enhanced self-renewal and colony formation abilities, and increased levels of stem cell marker genes. Mechanistically, DLK1-mediated effects on glioma cells involved increased and prolonged stabilization of hypoxia-inducible factor 2alpha, and inhibition of hypoxia-inducible factor 2alpha activity abolished effects of DLK1 in hypoxia. Forced expression of soluble DLK1 resulted in more aggressive tumor growth and shortened survival in a genetically engineered mouse model of glioma. Together, our data support DLK1 as a soluble mediator of glioma aggressiveness derived from the tumor microenvironment