Publications by authors named "Audrey Dumont"

12 Publications

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Regulation of kinesin-1 activity by the effectors PipB2 and SifA.

J Cell Sci 2020 05 14;133(9). Epub 2020 May 14.

Aix-Marseille Université, CNRS, INSERM, CIML, Marseille, France

is an intracellular bacterial pathogen. The formation of its replication niche, which is composed of a vacuole associated with a network of membrane tubules, depends on the secretion of a set of bacterial effector proteins whose activities deeply modify the functions of the eukaryotic host cell. By recruiting and regulating the activity of the kinesin-1 molecular motor, effectors PipB2 and SifA play an essential role in the formation of the bacterial compartments. In particular, they allow the formation of tubules from the vacuole and their extension along the microtubule cytoskeleton, and thus promote membrane exchanges and nutrient supply. We have developed and assays to better understand the specific role played by these two effectors in the recruitment and regulation of kinesin-1. Our results reveal a specific interaction between the two effectors and indicate that, contrary to what studies on infected cells suggested, interaction with PipB2 is sufficient to relieve the autoinhibition of kinesin-1. Finally, they suggest the involvement of other effectors in the control of the activity of this molecular motor.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.239863DOI Listing
May 2020

A passive physical model for DnaK chaperoning.

Phys Biol 2018 01 11;15(2):026003. Epub 2018 Jan 11.

Laboratoire de Chimie Bactérienne, Institut de Microbiologie de la Méditerranée, Aix-Marseille Université, CNRS, UMR 7283, 31 Chemin Joseph Aiguier, 13009 Marseille, France.

Almost all living organisms use protein chaperones with a view to preventing proteins from misfolding or aggregation either spontaneously or during cellular stress. This work uses a reaction-diffusion stochastic model to describe the dynamic localization of the Hsp70 chaperone DnaK in Escherichia coli cells during transient proteotoxic collapse characterized by the accumulation of insoluble proteins. In the model, misfolded ('abnormal') proteins are produced during alcoholic stress and have the propensity to aggregate with a polymerization-like kinetics. When aggregates diffuse more slowly they grow larger. According to Michaelis-Menten-type kinetics, DnaK has the propensity to bind with misfolded proteins or aggregates in order to catalyse refolding. To match experimental fluorescence microscopy data showing clusters of DnaK-GFP localized in multiple foci, the model includes spatial zones with local reduced diffusion rates to generate spontaneous assemblies of DnaK called 'foci'. Numerical simulations of our model succeed in reproducing the kinetics of DnaK localization experimentally observed. DnaK starts from foci, moves to large aggregates during acute stress, resolves those aggregates during recovery and finally returns to its initial punctate localization pattern. Finally, we compare real biological events with hypothetical repartitions of the protein aggregates or DnaK. We then notice that DnaK action is more efficient on protein aggregates than on protein homogeneously distributed.
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http://dx.doi.org/10.1088/1478-3975/aa9130DOI Listing
January 2018

Clinical Value of Natriuretic Peptides in Predicting Time to Dialysis in Stage 4 and 5 Chronic Kidney Disease Patients.

PLoS One 2016 22;11(8):e0159914. Epub 2016 Aug 22.

Service de Néphrologie, CHU Hôpitaux de Rouen, Rouen, France.

Background: Anticipating the time to renal replacement therapy (RRT) in chronic kidney disease (CKD) patients is an important but challenging issue. Natriuretic peptides are biomarkers of ventricular dysfunction related to poor outcome in CKD. We comparatively investigated the value of B-type natriuretic peptide (BNP) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) as prognostic markers for the risk of RRT in stage 4 and 5 CKD patients, and in foretelling all-cause mortality and major cardiovascular events within a 5-year follow-up period.

Methods: Baseline plasma BNP (Triage, Biosite) and NT-proBNP (Elecsys, Roche) were measured at inclusion. Forty-three patients were followed-up during 5 years. Kaplan-Meier analysis, with log-rank testing and hazard ratios (HR), were calculated to evaluate survival without RRT, cardiovascular events or mortality. The independent prognostic value of the biomarkers was estimated in separate Cox multivariate analysis, including estimated glomerular filtration rate (eGFR), creatininemia and comorbidities.

Results: During the first 12-month follow-up period, 16 patients started RRT. NT-proBNP concentration was higher in patients who reached endpoint (3221 ng/L vs 777 ng/L, p = 0.02). NT-proBNP concentration > 1345 ng/L proved significant predictive value on survival analysis for cardiovascular events (p = 0.04) and dialysis within 60 months follow-up (p = 0.008). BNP concentration > 140 ng/L was an independent predictor of RRT after 12 months follow-up (p<0.005), and of significant predictive value for initiation of dialysis within 60 months follow-up.

Conclusions: Our results indicate a prognostic value for BNP and NT-proBNP in predicting RRT in stage 4 and 5 CKD patients, regarding both short- and long-term periods. NT-proBNP also proved a value in predicting cardiovascular events. Natriuretic peptides could be useful predictive biomarkers for therapeutic guidance in CKD.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0159914PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4993513PMC
July 2017

Rapid and Specific Enrichment of Culturable Gram Negative Bacteria Using Non-Lethal Copper-Free Click Chemistry Coupled with Magnetic Beads Separation.

PLoS One 2015 10;10(6):e0127700. Epub 2015 Jun 10.

Aix Marseille Université, Laboratoire de Chimie Bactérienne (UMR 7283), Institut de Microbiologie de la Méditerranée (IMM), CNRS, 31 Chemin Joseph Aiguier-13402, Marseille, France.

Currently, identification of pathogenic bacteria present at very low concentration requires a preliminary culture-based enrichment step. Many research efforts focus on the possibility to shorten this pre-enrichment step which is needed to reach the minimal number of cells that allows efficient identification. Rapid microbiological controls are a real public health issue and are required in food processing, water quality assessment or clinical pathology. Thus, the development of new methods for faster detection and isolation of pathogenic culturable bacteria is necessary. Here we describe a specific enrichment technique for culturable Gram negative bacteria, based on non-lethal click chemistry and the use of magnetic beads that allows fast detection and isolation. The assimilation and incorporation of an analog of Kdo, an essential component of lipopolysaccharides, possessing a bio-orthogonal azido function (Kdo-N3), allow functionalization of almost all Gram negative bacteria at the membrane level. Detection can be realized through strain-promoted azide-cyclooctyne cycloaddition, an example of click chemistry, which interestingly does not affect bacterial growth. Using E. coli as an example of Gram negative bacterium, we demonstrate the excellent specificity of the technique to detect culturable E. coli among bacterial mixtures also containing either dead E. coli, or live B. subtilis (as a model of microorganism not containing Kdo). Finally, in order to specifically isolate and concentrate culturable E. coli cells, we performed separation using magnetic beads in combination with click chemistry. This work highlights the efficiency of our technique to rapidly enrich and concentrate culturable Gram negative bacteria among other microorganisms that do not possess Kdo within their cell envelope.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127700PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4465638PMC
June 2016

Identification of living Legionella pneumophila using species-specific metabolic lipopolysaccharide labeling.

Angew Chem Int Ed Engl 2014 Jan 20;53(5):1275-8. Epub 2014 Jan 20.

Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles du CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette (France).

Legionella pneumophila is a pathogenic bacterium involved in regular outbreaks characterized by a relatively high fatality rate and an important societal impact. Frequent monitoring of the presence of this bacterium in environmental water samples is necessary to prevent these epidemic events, but the traditional culture-based detection and identification method requires up to 10 days. Reported herein is a method allowing identification of Legionella pneumophila by metabolic lipopolysaccharide labeling which targets, for the first time, a precursor to monosaccharides that are specifically present within the O-antigen of the bacterium. This new approach allows easy detection of living Legionella pneumophila, while other Legionella species are not labeled.
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http://dx.doi.org/10.1002/anie.201309072DOI Listing
January 2014

Early sequence of events triggered by the interaction of Neisseria meningitidis with endothelial cells.

Cell Microbiol 2014 Jun 27;16(6):878-95. Epub 2013 Dec 27.

Université Paris Descartes, Faculté de Médecine Paris Descartes, Paris, F-75006, France; INSERM, U970, Paris Cardiovascular Research Center, Paris, F-75015, France.

Neisseria meningitidis is a bacterium responsible for severe sepsis and meningitis. Following type IV pilus-mediated adhesion to endothelial cells, bacteria proliferating on the cellular surface trigger a potent cellular response that enhances the ability of adhering bacteria to resist the mechanical forces generated by the blood flow. This response is characterized by the formation of numerous 100 nm wide membrane protrusions morphologically related to filopodia. Here, a high-resolution quantitative live-cell fluorescence microscopy procedure was designed and used to study this process. A farnesylated plasma membrane marker was first detected only a few seconds after bacterial contact, rapidly followed by actin cytoskeleton reorganization and bulk cytoplasm accumulation. The bacterial type IV pili-associated minor pilin PilV is necessary for the initiation of this cascade. Plasma membrane composition is a key factor as cholesterol depletion with methyl-β-cyclodextrin completely blocks the initiation of the cellular response. In contrast membrane deformation does not require the actin cytoskeleton. Strikingly, plasma membrane remodelling undermicrocolonies is also independent of common intracellular signalling pathways as cellular ATP depletion is not inhibitory. This study shows that bacteria-induced plasma membrane reorganization is a rapid event driven by a direct cross-talk between type IV pili and the plasma membrane rather than by the activation of an intracellular signalling pathway that would lead to actin remodelling.
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http://dx.doi.org/10.1111/cmi.12248DOI Listing
June 2014

Click-mediated labeling of bacterial membranes through metabolic modification of the lipopolysaccharide inner core.

Angew Chem Int Ed Engl 2012 Mar 9;51(13):3143-6. Epub 2012 Feb 9.

Aix Marseille Université, Laboratoire de Chimie, Bactérienne (UMR 7283), Institut de Microbiologie de la Méditerranée, CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille, France.

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http://dx.doi.org/10.1002/anie.201108127DOI Listing
March 2012

Posttranslational modification of pili upon cell contact triggers N. meningitidis dissemination.

Science 2011 Feb;331(6018):778-82

Ecole Polytechnique, Laboratoire des Mécanismes Réactionnels, Palaiseau F-91128, France.

The Gram-negative bacterium Neisseria meningitidis asymptomatically colonizes the throat of 10 to 30% of the human population, but throat colonization can also act as the port of entry to the blood (septicemia) and then the brain (meningitis). Colonization is mediated by filamentous organelles referred to as type IV pili, which allow the formation of bacterial aggregates associated with host cells. We found that proliferation of N. meningitidis in contact with host cells increased the transcription of a bacterial gene encoding a transferase that adds phosphoglycerol onto type IV pili. This unusual posttranslational modification specifically released type IV pili-dependent contacts between bacteria. In turn, this regulated detachment process allowed propagation of the bacterium to new colonization sites and also migration across the epithelium, a prerequisite for dissemination and invasive disease.
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http://dx.doi.org/10.1126/science.1200729DOI Listing
February 2011

SKIP, the host target of the Salmonella virulence factor SifA, promotes kinesin-1-dependent vacuolar membrane exchanges.

Traffic 2010 Jul 6;11(7):899-911. Epub 2010 Apr 6.

Centre d'Immunologie de Marseille-Luminy, CNRS UMR 6102, INSERM U631, Université de la Méditerranée, Parc Scientifique de Luminy, Case 906-13288 Marseille Cedex 9, France.

In Salmonella-infected cells, the bacterial effector SifA forms a functional complex with the eukaryotic protein SKIP (SifA and kinesin-interacting protein). The lack of either partner has important consequences on the intracellular fate and on the virulence of this pathogen. In addition to SifA, SKIP binds the microtubule-based motor kinesin-1. Yet the absence of SifA or SKIP results in an unusual accumulation of kinesin-1 on the bacterial vacuolar membrane. To understand this apparent contradiction, we investigated the interaction between SKIP and kinesin-1 and the function of this complex. We show that the C-terminal RUN (RPIP8, UNC-14 and NESCA) domain of SKIP interacted specifically with the tetratricopeptide repeat (TPR) domain of the kinesin light chain. Overexpression of SKIP induced a microtubule- and kinesin-1-dependent anterograde movement of late endosomal/lysosomal compartments. In infected cells, SifA contributed to the fission of vesicles from the bacterial vacuole and the SifA/SKIP complex was required for the formation and/or the anterograde transport of kinesin-1-enriched vesicles. These observations reflect the role of SKIP as a linker and/or an activator for kinesin-1.
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http://dx.doi.org/10.1111/j.1600-0854.2010.01069.xDOI Listing
July 2010

Interaction between the SifA virulence factor and its host target SKIP is essential for Salmonella pathogenesis.

J Biol Chem 2009 Nov 28;284(48):33151-60. Epub 2009 Sep 28.

Centre d'Immunologie de Marseille-Luminy, CNRS UMR 6102, INSERM U631, Université de la Méditerranée, Parc Scientifique de Luminy, Case 906, 13288 Marseille Cedex 9.

SifA is a Salmonella effector that is translocated into infected cells by the pathogenicity island 2-encoded type 3 secretion system. SifA is a critical virulence factor. Previous studies demonstrated that, upon translocation, SifA binds the pleckstrin homology motif of the eukaryotic host protein SKIP. In turn, the SifA-SKIP complex regulates the mobilization of the molecular motor kinesin-1 on the bacterial vacuole. SifA exhibits multiple domains containing functional motifs. Here we performed a molecular dissection and a mutational study of SifA to evaluate the relative contribution of the different domains to SifA functions. Biochemical and crystallographic analysis confirmed that the N-terminal domain of SifA is sufficient to interact with the pleckstrin homology domain of SKIP, forming a 1:1 complex with a micromolar dissociation constant. Mutation of the tryptophan residue in the WXXXE motif, which has been proposed to mimic active form of GTPase, deeply affected the stability and the translocation of SifA while mutations of the glutamic residue had no functional impact. A SifA L130D mutant that does not bind SKIP showed a DeltasifA-like phenotype both in infected cells and in the mouse model of infection. We concluded that the WXXXE motif is essential for maintaining the tertiary structure of SifA, the functions of which require the interaction with the eukaryotic protein SKIP.
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http://dx.doi.org/10.1074/jbc.M109.034975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785157PMC
November 2009

Analysis of kinesin accumulation on Salmonella-containing vacuoles.

Methods Mol Biol 2007 ;394:275-87

Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Méditerranée, Parc Scientifique de Luminy, France.

Salmonella enterica is an intracellular bacterial pathogen that causes gastroenteritis and typhoid fever. Inside host cells, the bacterium is enclosed in a membrane bound compartment, the Salmonella-containing vacuole (SCV). Intracellular replication of Salmonella requires the translocation of effector proteins into the host cytosol. The SifA effector protein is important for the membrane stability of the SCV. Recently, we have shown that the Salmonella sifA- mutant presents on its vacuole an important accumulation of kinesin-1, a molecular motor involved in the plus-end-directed transport of various organelles. Kinesin-1 is not recruited on SCVs of mutants that do not translocate effector proteins. This indicates that SifA is a negative regulator of the recruitment of this molecular motor and reveals the existence of another effector that recruits kinesin-1. This chapter describes techniques that are used to screen by immunofluorescence microscopy the accumulation of kinesin-1 on strains of Salmonella carrying multiple mutations.
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http://dx.doi.org/10.1007/978-1-59745-512-1_13DOI Listing
April 2008

The Salmonella effector protein PipB2 is a linker for kinesin-1.

Proc Natl Acad Sci U S A 2006 Sep 25;103(36):13497-502. Epub 2006 Aug 25.

Centre d'Immunologie de Marseille-Luminy, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université de la Méditerranée, Parc Scientifique de Luminy, Marseille, France.

Understanding the mechanisms of Salmonella virulence is an important challenge. The capacity of this intracellular bacterial pathogen to cause diseases depends on the expression of virulence factors including the second type III secretion system (TTSS-2), which is used to translocate into the eukaryotic cytosol a set of effector proteins that divert the biology of the host cell and shape the bacterial replicative niche. Yet little is known about the eukaryotic functions affected by individual Salmonella effectors. Here we report that the TTSS-2 effector PipB2 interacts with the kinesin light chain, a subunit of the kinesin-1 motor complex that drives anterograde transport along microtubules. Translocation of PipB2 is both necessary and sufficient for the recruitment of kinesin-1 to the membrane of the Salmonella-containing vacuole. In vivo, PipB2 contributes to the attenuation of Salmonella mutant strains in mice. Taken together, our data indicate that the TTSS-2-mediated fine-tuning of kinesin-1 activity associated with the bacterial vacuole is crucial for the virulence of Salmonella.
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http://dx.doi.org/10.1073/pnas.0605443103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1569191PMC
September 2006