Cycle Inhibiting Factors (Cifs): Cyclomodulins That Usurp the Ubiquitin-Dependent Degradation Pathway of Host Cells

Cycle inhibiting factors (Cifs) are type III secreted effectors produced by diverse pathogenic bacteria. Cifs are “cyclomodulins” that inhibit the eukaryotic host cell cycle and also hijack other key cellular processes such as those controlling the actin network and apoptosis. This review summarizes current knowledge on Cif since its first characterization in enteropathogenic Escherichia coli, the identification of several xenologues in distant pathogenic bacteria, to its structure elucidation and the recent deciphering of its mode of action. Cif impairs the host ubiquitin proteasome system through deamidation of ubiquitin or the ubiquitin-like protein NEDD8 that regulates Cullin-Ring-ubiquitin Ligase (CRL) complexes. The hijacking of the ubiquitin-dependent degradation pathway of host cells results in the modulation of various cellular functions such as epithelium renewal, apoptosis and immune response. Cif is therefore a powerful weapon in the continuous arm race that characterizes host-bacteria interactions

Pathogenic Bacteria Target NEDD8-Conjugated Cullins to Hijack Host-Cell Signaling Pathways

The cycle inhibiting factors (Cif), produced by pathogenic bacteria isolated from vertebrates and invertebrates, belong to a family of molecules called cyclomodulins that interfere with the eukaryotic cell cycle. Cif blocks the cell cycle at both the G1/S and G2/M transitions by inducing the stabilization of cyclin-dependent kinase inhibitors p21waf1 and p27kip1. Using yeast two-hybrid screens, we identified the ubiquitin-like protein NEDD8 as a target of Cif. Cif co-compartmentalized with NEDD8 in the host cell nucleus and induced accumulation of NEDD8-conjugated cullins. This accumulation occurred early after cell infection and correlated with that of p21 and p27. Co-immunoprecipitation revealed that Cif interacted with cullin-RING ubiquitin ligase complexes (CRLs) through binding with the neddylated forms of cullins 1, 2, 3, 4A and 4B subunits of CRL. Using an in vitro ubiquitylation assay, we demonstrate that Cif directly inhibits the neddylated CUL1-associated ubiquitin ligase activity. Consistent with this inhibition and the interaction of Cif with several neddylated cullins, we further observed that Cif modulates the cellular half-lives of various CRL targets, which might contribute to the pathogenic potential of diverse bacteria

Constraints on the structure and seasonal variations of Triton's atmosphere from the 5 October 2017 stellar occultation and previous observations

Context. A stellar occultation by Neptune's main satellite, Triton, was observed on 5 October 2017 from Europe, North Africa, and the USA. We derived 90 light curves from this event, 42 of which yielded a central flash detection. Aims. We aimed at constraining Triton's atmospheric structure and the seasonal variations of its atmospheric pressure since the Voyager 2 epoch (1989). We also derived the shape of the lower atmosphere from central flash analysis. Methods. We used Abel inversions and direct ray-tracing code to provide the density, pressure, and temperature profiles in the altitude range similar to 8 km to similar to 190 km, corresponding to pressure levels from 9 mu bar down to a few nanobars. Results. (i) A pressure of 1.18 +/- 0.03 mu bar is found at a reference radius of 1400 km (47 km altitude). (ii) A new analysis of the Voyager 2 radio science occultation shows that this is consistent with an extrapolation of pressure down to the surface pressure obtained in 1989. (iii) A survey of occultations obtained between 1989 and 2017 suggests that an enhancement in surface pressure as reported during the 1990s might be real, but debatable, due to very few high S/N light curves and data accessible for reanalysis. The volatile transport model analysed supports a moderate increase in surface pressure, with a maximum value around 2005-2015 no higher than 23 mu bar. The pressures observed in 1995-1997 and 2017 appear mutually inconsistent with the volatile transport model presented here. (iv) The central flash structure does not show evidence of an atmospheric distortion. We find an upper limit of 0.0011 for the apparent oblateness of the atmosphere near the 8 km altitude.J.M.O. acknowledges financial support from the Portuguese Foundation for Science and Technology (FCT) and the European Social Fund (ESF) through the PhD grant SFRH/BD/131700/2017. The work leading to these results has received funding from the European Research Council under the European Community's H2020 2014-2021 ERC grant Agreement nffi 669416 "Lucky Star". We thank S. Para who supported some travels to observe the 5 October 2017 occultation. T.B. was supported for this research by an appointment to the National Aeronautics and Space Administration (NASA) Post-Doctoral Program at the Ames Research Center administered by Universities Space Research Association (USRA) through a contract with NASA. We acknowledge useful exchanges with Mark Gurwell on the ALMA CO observations. This work has made use of data from the European Space Agency (ESA) mission Gaia (https://www.cosmos.esa.int/gaia), processed by the Gaia Data Processing and Analysis Consortium (DPAC, https://www.cosmos.esa.int/web/gaia/dpac/consortium).Funding for the DPAC has been provided by national institutions, in particular the institutions participating in the Gaia Multilateral Agreement. J.L.O., P.S.-S., N.M. and R.D. acknowledge financial support from the State Agency for Research of the Spanish MCIU through the "Center of Excellence Severo Ochoa" award to the Instituto de Astrofisica de Andalucia (SEV-2017-0709), they also acknowledge the financial support by the Spanish grant AYA-2017-84637-R and the Proyecto de Excelencia de la Junta de Andalucia J.A. 2012-FQM1776. The research leading to these results has received funding from the European Union's Horizon 2020 Research and Innovation Programme, under Grant Agreement no. 687378, as part of the project "Small Bodies Near and Far" (SBNAF). P.S.-S. acknowledges financial support by the Spanish grant AYA-RTI2018-098657-J-I00 "LEO-SBNAF". The work was partially based on observations made at the Laboratorio Nacional de Astrofisica (LNA), Itajuba-MG, Brazil. The following authors acknowledge the respective CNPq grants: F.B.-R. 309578/2017-5; R.V.-M. 304544/2017-5, 401903/2016-8; J.I.B.C. 308150/2016-3 and 305917/2019-6; M.A. 427700/20183, 310683/2017-3, 473002/2013-2. This study was financed in part by the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior -Brasil (CAPES) -Finance Code 001 and the National Institute of Science and Technology of the e-Universe project (INCT do e-Universo, CNPq grant 465376/2014-2). G.B.R. acknowledges CAPES-FAPERJ/PAPDRJ grant E26/203.173/2016 and CAPES-PRINT/UNESP grant 88887.571156/2020-00, M.A. FAPERJ grant E26/111.488/2013 and A.R.G.Jr. FAPESP grant 2018/11239-8. B.E.M. thanks CNPq 150612/2020-6 and CAPES/Cofecub-394/2016-05 grants. Part of the photometric data used in this study were collected in the frame of the photometric observations with the robotic and remotely controlled telescope at the University of Athens Observatory (UOAO; Gazeas 2016). The 2.3 m Aristarchos telescope is operated on Helmos Observatory by the Institute for Astronomy, Astrophysics, Space Applications and Remote Sensing of the National Observatory of Athens. Observations with the 2.3 m Aristarchos telescope were carried out under OPTICON programme. This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 730890. This material reflects only the authors views and the Commission is not liable for any use that may be made of the information contained therein. The 1. 2m Kryoneri telescope is operated by the Institute for Astronomy, Astrophysics, Space Applications and Remote Sensing of the National Observatory of Athens. The Astronomical Observatory of the Autonomous Region of the Aosta Valley (OAVdA) is managed by the Fondazione Clement Fillietroz-ONLUS, which is supported by the Regional Government of the Aosta Valley, the Town Municipality of Nus and the "Unite des Communes valdotaines Mont-Emilius". The 0.81 m Main Telescope at the OAVdA was upgraded thanks to a Shoemaker NEO Grant 2013 from The Planetary Society. D.C. and J.M.C. acknowledge funds from a 2017 'Research and Education' grant from Fondazione CRT-Cassa di Risparmio di Torino. P.M. acknowledges support from the Portuguese Fundacao para a Ciencia e a Tecnologia ref. PTDC/FISAST/29942/2017 through national funds and by FEDER through COMPETE 2020 (ref. POCI010145 FEDER007672). F.J. acknowledges Jean Luc Plouvier for his help. S.J.F. and C.A. would like to thank the UCL student support observers: Helen Dai, Elise Darragh-Ford, Ross Dobson, Max Hipperson, Edward Kerr-Dineen, Isaac Langley, Emese Meder, Roman Gerasimov, Javier Sanjuan, and Manasvee Saraf. We are grateful to the CAHA, OSN and La Hita Observatory staffs. This research is partially based on observations collected at Centro Astronomico HispanoAleman (CAHA) at Calar Alto, operated jointly by Junta de Andalucia and Consejo Superior de Investigaciones Cientificas (IAA-CSIC). This research was also partially based on observation carried out at the Observatorio de Sierra Nevada (OSN) operated by Instituto de Astrofisica de Andalucia (CSIC). This article is also based on observations made with the Liverpool Telescope operated on the island of La Palma by Liverpool John Moores University in the Spanish Observatorio del Roque de los Muchachos of the Instituto de Astrofisica de Canarias with financial support from the UK Science and Technology Facilities Council. Partially based on observations made with the Tx40 and Excalibur telescopes at the Observatorio Astrofisico de Javalambre in Teruel, a Spanish Infraestructura Cientifico-Tecnica Singular (ICTS) owned, managed and operated by the Centro de Estudios de Fisica del Cosmos de Aragon (CEFCA). Tx40 and Excalibur are funded with the Fondos de Inversiones de Teruel (FITE). A.R.R. would like to thank Gustavo Roman for the mechanical adaptation of the camera to the telescope to allow for the observation to be recorded. R.H., J.F.R., S.P.H. and A.S.L. have been supported by the Spanish projects AYA2015-65041P and PID2019-109467GB-100 (MINECO/FEDER, UE) and Grupos Gobierno Vasco IT1366-19. Our great thanks to Omar Hila and their collaborators in Atlas Golf Marrakech Observatory for providing access to the T60cm telescope. TRAPPIST is a project funded by the Belgian Fonds (National) de la Recherche Scientifique (F.R.S.-FNRS) under grant PDR T.0120.21. TRAPPIST-North is a project funded by the University of Liege, and performed in collaboration with Cadi Ayyad University of Marrakesh. E.J. is a FNRS Senior Research Associate

Rôle des génotoxines produites par des bactéries du microbiote dans le cancer colorectal

Numerous studies support a role for the intestinal microbiota in colorectal tumorigenesis. Recent results have suggested that bacteria of the microbiota could be associated with the presence of tumours. Some can be directly genotoxic : as we showed, strains of Escherichia coli, an ubiquitous member of the colon flora, synthesize a genotoxin called colibactin.These bacteria induce DNA double strand breaks in intestinal cells and trigger chromosomal instability, gene mutations and cell transformation. Moreover it was recently shown that colibactin-producing E. coli promote colorectal cancer in a murine IL10-/- model. Thus long-term colonization of the colon with rogue commensal bacteria capable of causing chronic DNA-damage could contribute to sporadic colorectal cancer development.De récents travaux suggèrent que le microbiote intestinal pourrait jouer un rôle important dans la carcinogenèse colorectale. Des études ont mis en évidence que des bactéries commensales du microbiote pouvaient êtreassociées à la présence de tumeurs. Certaines peuvent être directement génotoxiques : c’est le cas de Escherichia coli, une des bactéries ubiquitaires anaérobies facultatives prédominantes de la flore du côlon. Nous avons ainsi découvert que certaines souches de E. coli synthétisent une génotoxine, la colibactine. Ces souches induisent des cassures double brin de l’ADN des cellules de la muqueuse intestinale et déclenchent une instabilité chromosomique, des mutations géniques et la transformation cellulaire, moteurs fondamentaux de la carcinogenèse. Il a été démontré récemment que ces E. coli produisant la colibactine ont un effet promoteurdans un modèle murin IL10-/- de cancérogenèse colorectale. La présence dans le microbiote intestinal de souches bactériennes qui produisent des toxines lésant l’ADN des cellules de l’hôte pourrait donc constituerun facteur prédisposant au développement de la forme sporadique du cancer colorectal

Role du système Csr dans la nutrition carbonée et dans le controle du métabolisme central d'Escherichia coli K-12 MG1655 et Nissle 1917

L implantation d'Escherichia coli dans l intestin résulte de stratégies adaptatives globales permettant à la bactérie de survivre face aux changements de conditions environnementales. Sur le plan métabolique, la colonisation de l intestin par E. coli parait associée à la disponibilité de sources de carbone préférentielles glycolytiques, alors que la maintenance et la persistance reposent sur sa capacité à utiliser différents substrats alternatifs (principalement gluconéogéniques) lorsque les substrats préférentiels deviennent limitant. Cette activation des processus gluconéogéniques, qui implique une réorganisation fonctionnelle complète du métabolisme, est associée in situ à de profonds remaniements physiologiques (perte de motilité, formation de biofilms, etc) nécessaires à la persistance d'E. coli dans l intestin. Ces différents processus sont coordonnés par des réseaux de régulation particulièrement complexes, dont le système Csr (Carbon storage regulator), un des principaux régulateur post-transcriptionnel d'E. coli. Lors de ces travaux, nous avons analysé le rôle du système Csr dans la nutrition carbonée et le contrôle du métabolisme central d'E. coli sur des sources de carbone supportant sa croissance dans l'intestin et représentatives des principales voies de son métabolisme central (glycolyse, voie des pentoses phosphate, voie d'Entner-Doudoroff cycle de Krebs). Cette étude a été réalisée chez deux souches d'E. coli présentant des capacités d'implantation distinctes : la souche de laboratoire K12 MG1655, et la souche Nissle 1917, un excellent colonisateur appartenant au groupe phylogénétique B2. Une analyse détaillée du fonctionnement métabolique par des approches systémiques quantitatives haut-débit (métabolomique et analyse des flux métaboliques par marquage isotopique) a été mise en place. Elle a été exploitée pour caractériser finement le comportement métabolique de souches sauvages et de mutants du système Csr d'E. coli sur différentes sources de carbone, identifier des caractères métaboliques propres à chaque souche, et étendre le rôle du système Csr dans la nutrition carbonée et dans le contrôle du métabolisme central d'E. coli. Nos résultats démontrent que i) Csr favorise l'utilisation d'un large spectre de sources de carbone aussi bien glycolytiques que gluconéogéniques, ii) Csr contrôle un nombre de voies métaboliques plus important que ce que l'on pourrait attendre à partir de ses cibles identifiées, et iii) que le contrôle global exercé par le système Csr sur le fonctionnement du métabolisme central dépend de la source de carbone. Un rôle du système Csr dans le contrôle du métabolisme redox (production de NADH et de NADPH) et énergétique (production d'ATP), non reporté à ce jour, est également démontré. Enfin, nos résultats suggèrent un rôle de Csr dans le contrôle de la balance anabolisme-catabolisme d'E. coli. Ces travaux renforcent le rôle potentiel du système Csr dans l'adaptation d'E. coli face aux changements de conditions environnementales.The implantation of Escherichia coli in the gut results from global adaptive strategies that allow the bacteria to survive in the changing environment of the intestine. At the metabolic level, recent findings indicate that colonisation is mainly related to the utilization of sugars and sugar derivatives through glycolytic pathways. In contrast, persistence of E. coli in the gut is supported by less favorable substrates, including small organic acids. The use of the latter compounds requires activation of gluconeogenic pathways, and efficient switching between glycolytic and gluconeogenic carbon sources is likely to be a major feature of successful adaptation to life in the intestine. These adaptive processes are controlled by highly sophisticated regulatory networks, such as the Csr (carbon storage regulator) system which is the main post-transcriptional regulator in E. coli. Csr was found to control a broad range of phenotypes allowing E. coli to successfully implant and persist in the gut, such as biofilm formation, motility as well as many functions involved in carbon nutrition, including glycolysis, gluconeogenesis, acetate and glycogen metabolism. Although Csr is likely to play an important role in the adaptation of bacteria to the nutritional context of the host, it is poorly understood sofar. In this work, we investigate the role of the Csr system in the control of E. coli metabolism on physiologically-relevant carbon sources representative of the main glycolytic (Entner-Doudoroff pathway, pentose phosphate pathway, glycolysis) and gluconeogenic pathways of E. coli. This work was carried out on two E. coli strains with distinct implantation capabilities : the K12 MG1655 laboratory strain and the Nissle 1917strain, an efficient colonizer of the gut belonging to the highly competitive B2phylogenetic group. First, we designed a complete methodology (metabolomics and 13C-metabolic flux analysis) for quantitative, system-level investigations of the actual operation of E. coli metabolism. Then, we performed detailed, system-level investigations of wild-type strains and Csr mutants. This work provides valuable information regarding systemic properties of E. coli metabolism, and identifies metabolic specificities of the Nissle 1917strain likely involved in its competitiveness in the gut. The role of Csr appears to be qualitatively and quantitatively the same in both K12 MG1655 and Nissle 1917 strains. We show that i) Csr enhances the utilisation of a broad spectrum of glycolytic and gluconeogenic carbon sources, ii) Csr controls a range of metabolic pathways wider than expected from its known target enzymes, and iii) the actual impact of the Csr system on the central metabolism of E. coli depends on the carbon source. We also demonstrate that Csr controls energy and redox metabolism in E. coli. Csr enhances the production of ATP and of reduced cofactors (NADH and NADPH), and we suggest that it also may control the catabolism-anabolism balance in E. coli. Finally, our results reinforce the potential role of the Csr system in the global adaptation of the bacterium to the gut environment.TOULOUSE-INSA-Bib. electronique (315559905) / SudocSudocFranceF

Effets génotoxiques des souches de Escherichia coli produisant la colibactine

TOULOUSE3-BU Sciences (315552104) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Interaction of Eschirichia coli producing cytotoxic necrotizing factor with hela epithelial cells

chap. 58International audienc

Vaccination orale contre les infections par les REPEC grâce à une souche bactérienne vivante atténuée double mutante

National audienc

Mitotic block and delayed lethality in HeLa epithelial cells exposed to Escherichia coli BM2-1 producing cytotoxic necrotizing factor type 1

The cytopathic effect (CPE) of Escherichia coli producing cytotoxic necrotizing factor type 1 (CNF1) was investigated by using a human epithelial cell (HeLa) model of infection with CNF1-producing E. coli BM2-1. This strain was shown to bind loosely, but massively, to HeLa cells. A 4-h interaction between bacteria and eukaryotic cells triggered the delayed appearance of a progressive dose-dependent CPE characterized by (i) intense swelling of cells accompanied by the formation of a dense network of actin stress fibers, (ii) inhibition of cell division due to a complete block in the G2 phase of the cell cycle, and (iii) nucleus swelling and chromatin fragmentation. These alterations resulted in cell death starting about 5 days after interaction. The absence of multinucleation clearly distinguished the CPE from the effect produced by cell-free culture supernatants of infected cells nor prevented by a CNF1-neutralizing antiserum. Pathogenicity was completely abolished after Tn5::phoA insertion mutagenesis in the cnf-1 structural gene but not restored by trans complementation with a recombinant plasmid containing intact cnf-1 and its promoter. These results suggest that a gene downstream of cnf-1, essential to the induction of the CPE, was affected by the mutation. On the other hand, transformation of the wild-type strain BM2-1 with the same recombinant plasmid leads to a significant increase in both CNF1 activity and CPE, demonstrating the direct contribution of CNF1 to the CPE. In conclusion, the pathogenicity of E. coli BM2-1 for HeLa cells results from a complex interaction involving cnf-1 and associated genes and possibly requiring a preliminary step of binding of bacterial organisms to target cells

Rôle de la régulation post-transcriptionnelle dans le contrôle du métabolisme carboné

International audienceLe métabolisme est l’objet d’une intense régulation dont les composantes métaboliques et transcriptionnelles sont largement étudiées. En revanche, le rôle de la régulation posttranscriptionnelle dans le contrôle du métabolisme carboné a été très peu caractérisé. La connaissance de ces mécanismes est pourtant essentielle pour relier l’expression du génome au comportement métabolique final. Dans ce travail, nous avons analysé le rôle du principal systèmede régulation post-transcriptionnelle chez la bactérie Escherichia coli, le système Csr (pour Carbon Storage Regulator), dans la nutrition carbonée et le contrôle du métabolisme central de la bactérie.Pour cela nous avons analysé les capacités de croissance de mutants affectés pour les différents composants du système Csr (deux protéines CsrA 51 et CsrD, et deux petits ARN non codants CsrB and CsrC) pour des sources de carbone représentatives de la principale niche écologique d’E. coli (intestin). Puis nous avons analysé en détail l’impact de ces mutations sur le métabolome et le fluxome de la bactérie. Nos résultats montrent que la protéine CsrA, qui est le constituant clé du système Csr, est un déterminant important de l’’utilisation de ces sources de carbone physiologiques. L’analyse fonctionnelle du métabolisme des mutants sur glucose et gluconate meten évidence une réorganisation importante du métabolisme carboné central, indiquant un rôle significatif du système Csr dans le contrôle du métabolisme carboné et énergétique (ATP et rédox).De plus, la nature et l’importance de ces réorganisations dépendent de la source de carbone. L’ensemble de ces résultats suggère un rôle significatif du système Csr dans l’adaptation métabolique aux conditions de vie que rencontre E. coli dans l’intestin