Publications by authors named "Aline Rifflet"

12 Publications

  • Page 1 of 1

A Secreted NlpC/P60 Endopeptidase from Photobacterium damselae subsp. Cleaves the Peptidoglycan of Potentially Competing Bacteria.

mSphere 2021 02 3;6(1). Epub 2021 Feb 3.

Fish Immunology and Vaccinology Group, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal

Peptidoglycan (PG) is a major component of the bacterial cell wall, forming a mesh-like structure enwrapping the bacteria that is essential for maintaining structural integrity and providing support for anchoring other components of the cell envelope. PG biogenesis is highly dynamic and requires multiple enzymes, including several hydrolases that cleave glycosidic or amide bonds in the PG. This work describes the structural and functional characterization of an NlpC/P60-containing peptidase from subsp. (), a Gram-negative bacterium that causes high mortality of warm-water marine fish with great impact for the aquaculture industry. PnpA ( lpC-like rotein ) has a four-domain structure with a hydrophobic and narrow access to the catalytic center and specificity for the γ-d-glutamyl--diaminopimelic acid bond. However, PnpA does not cleave the PG of or PG of several Gram-negative and Gram-positive bacterial species. Interestingly, it is secreted by the type II secretion system and degrades the PG of and This suggests that PnpA is used by to gain an advantage over bacteria that compete for the same resources or to obtain nutrients in nutrient-scarce environments. Comparison of the muropeptide composition of PG susceptible and resistant to the catalytic activity of PnpA showed that the global content of muropeptides is similar, suggesting that susceptibility to PnpA is determined by the three-dimensional organization of the muropeptides in the PG. Peptidoglycan (PG) is a major component of the bacterial cell wall formed by long chains of two alternating sugars interconnected by short peptides, generating a mesh-like structure that enwraps the bacterial cell. Although PG provides structural integrity and support for anchoring other components of the cell envelope, it is constantly being remodeled through the action of specific enzymes that cleave or join its components. Here, it is shown that subsp. , a bacterium that causes high mortality in warm-water marine fish, produces PnpA, an enzyme that is secreted into the environment and is able to cleave the PG of potentially competing bacteria, either to gain a competitive advantage and/or to obtain nutrients. The specificity of PnpA for the PG of some bacteria and its inability to cleave others may be explained by differences in the structure of the PG mesh and not by different muropeptide composition.
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http://dx.doi.org/10.1128/mSphere.00736-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7860986PMC
February 2021

NOD1 sensing of house dust mite-derived microbiota promotes allergic experimental asthma.

J Allergy Clin Immunol 2021 Jan 25. Epub 2021 Jan 25.

University of Lille, Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale, Centre Hospitalier Universitaire Lille, Institut Pasteur de Lille, U1019-Unite Mixte de Recherche (UMR) 9017-Centre d'Infection et d'Immunité de Lille, Lille, France. Electronic address:

Background: Asthma severity has been linked to exposure to gram-negative bacteria from the environment that are recognized by NOD1 receptor and are present in house dust mite (HDM) extracts. NOD1 polymorphism has been associated with asthma.

Objective: We sought to evaluate whether either host or HDM-derived microbiota may contribute to NOD1-dependent disease severity.

Methods: A model of HDM-induced experimental asthma was used and the effect of NOD1 deficiency was evaluated. Contribution of host microbiota was evaluated by fecal transplantation. Contribution of HDM-derived microbiota was assessed by 16S ribosomal RNA sequencing, mass spectrometry analysis, and peptidoglycan depletion of the extracts.

Results: In this model, loss of the bacterial sensor NOD1 and its adaptor RIPK2 improved asthma features. Such inhibitory effect was not related to dysbiosis caused by NOD1 deficiency, as shown by fecal transplantation of Nod1-deficient microbiota to wild-type germ-free mice. The 16S ribosomal RNA gene sequencing and mass spectrometry analysis of HDM allergen, revealed the presence of some muropeptides from gram-negative bacteria that belong to the Bartonellaceae family. While such HDM-associated muropeptides were found to activate NOD1 signaling in epithelial cells, peptidoglycan-depleted HDM had a decreased ability to instigate asthma in vivo.

Conclusions: These data show that NOD1-dependent sensing of HDM-associated gram-negative bacteria aggravates the severity of experimental asthma, suggesting that inhibiting the NOD1 signaling pathway may be a therapeutic approach to treating asthma.
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http://dx.doi.org/10.1016/j.jaci.2020.12.649DOI Listing
January 2021

Effect of gut microbiota on depressive-like behaviors in mice is mediated by the endocannabinoid system.

Nat Commun 2020 12 11;11(1):6363. Epub 2020 Dec 11.

Perception and Memory Unit, CNRS UMR3571, Institut Pasteur, Paris, France.

Depression is the leading cause of disability worldwide. Recent observations have revealed an association between mood disorders and alterations of the intestinal microbiota. Here, using unpredictable chronic mild stress (UCMS) as a mouse model of depression, we show that UCMS mice display phenotypic alterations, which could be transferred from UCMS donors to naïve recipient mice by fecal microbiota transplantation. The cellular and behavioral alterations observed in recipient mice were accompanied by a decrease in the endocannabinoid (eCB) signaling due to lower peripheral levels of fatty acid precursors of eCB ligands. The adverse effects of UCMS-transferred microbiota were alleviated by selectively enhancing the central eCB or by complementation with a strain of the Lactobacilli genus. Our findings provide a mechanistic scenario for how chronic stress, diet and gut microbiota generate a pathological feed-forward loop that contributes to despair behavior via the central eCB system.
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http://dx.doi.org/10.1038/s41467-020-19931-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732982PMC
December 2020

Spatiotemporal analysis of mycolactone distribution in vivo reveals partial diffusion in the central nervous system.

PLoS Negl Trop Dis 2020 12 2;14(12):e0008878. Epub 2020 Dec 2.

Immunobiology of Infection Unit, Institut Pasteur, INSERM U1221, Paris, France.

Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU) disease, is unique amongst human pathogens in its capacity to produce a lipid toxin called mycolactone. While previous studies have demonstrated that bacterially-released mycolactone diffuses beyond infection foci, the spatiotemporal distribution of mycolactone remained largely unknown. Here, we used the zebrafish model to provide the first global kinetic analysis of mycolactone's diffusion in vivo, and multicellular co-culture systems to address the critical question of the toxin's access to the brain. Zebrafish larvae were injected with a fluorescent-derivative of mycolactone to visualize the in vivo diffusion of the toxin from the peripheral circulation. A rapid, body-wide distribution of mycolactone was observed, with selective accumulation in tissues near the injection site and brain, together with an important excretion through the gastro-intestinal tract. Our conclusion that mycolactone reached the central nervous system was reinforced by an in cellulo model of human blood brain barrier and a mouse model of M. ulcerans-infection. Here we show that mycolactone has a broad but heterogenous profile of distribution in vivo. Our investigations in vitro and in vivo support the view that a fraction of bacterially-produced mycolactone gains access to the central nervous system. The relative persistence of mycolactone in the bloodstream suggests that assays of circulating mycolactone are relevant for BU disease monitoring and treatment optimization.
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http://dx.doi.org/10.1371/journal.pntd.0008878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710047PMC
December 2020

A peptide of a type I toxin-antitoxin system induces morphological transformation from spiral shape to coccoids.

Proc Natl Acad Sci U S A 2020 12 23;117(49):31398-31409. Epub 2020 Nov 23.

Unité Pathogenèse de Helicobacter, CNRS UMR 2001, Département de Microbiologie, Institut Pasteur, 75724 Paris Cedex 15, France;

Toxin-antitoxin systems are found in many bacterial chromosomes and plasmids with roles ranging from plasmid stabilization to biofilm formation and persistence. In these systems, the expression/activity of the toxin is counteracted by an antitoxin, which, in type I systems, is an antisense RNA. While the regulatory mechanisms of these systems are mostly well defined, the toxins' biological activity and expression conditions are less understood. Here, these questions were investigated for a type I toxin-antitoxin system (AapA1-IsoA1) expressed from the chromosome of the human pathogen We show that expression of the AapA1 toxin in causes growth arrest associated with rapid morphological transformation from spiral-shaped bacteria to round coccoid cells. Coccoids are observed in patients and during in vitro growth as a response to different stress conditions. The AapA1 toxin, first molecular effector of coccoids to be identified, targets inner membrane without disrupting it, as visualized by cryoelectron microscopy. The peptidoglycan composition of coccoids is modified with respect to spiral bacteria. No major changes in membrane potential or adenosine 5'-triphosphate (ATP) concentration result from AapA1 expression, suggesting coccoid viability. Single-cell live microscopy tracking the shape conversion suggests a possible association of this process with cell elongation/division interference. Oxidative stress induces coccoid formation and is associated with repression of the antitoxin promoter and enhanced processing of its transcript, leading to an imbalance in favor of AapA1 toxin expression. Our data support the hypothesis of viable coccoids with characteristics of dormant bacteria that might be important in infections refractory to treatment.
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http://dx.doi.org/10.1073/pnas.2016195117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7733810PMC
December 2020

Peptidoglycan analysis reveals that synergistic deacetylase activity in vegetative impacts the host response.

J Biol Chem 2020 12 25;295(49):16785-16796. Epub 2020 Sep 25.

Université Paris-Saclay, INRAE, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France. Electronic address:

is an anaerobic and spore-forming bacterium responsible for 15-25% of postantibiotic diarrhea and 95% of pseudomembranous colitis. Peptidoglycan is a crucial element of the bacterial cell wall that is exposed to the host, making it an important target for the innate immune system. The peptidoglycan is largely -deacetylated on its glucosamine (93% of muropeptides) through the activity of enzymes known as -deacetylases, and this -deacetylation modulates host-pathogen interactions, such as resistance to the bacteriolytic activity of lysozyme, virulence, and host innate immune responses. genome analysis showed that 12 genes potentially encode -deacetylases; however, which of these -deacetylases are involved in peptidoglycan deacetylation remains unknown. Here, we report the enzymes responsible for peptidoglycan -deacetylation and their respective regulation. Through peptidoglycan analysis of several mutants, we found that the deacetylases PdaV and PgdA act in synergy. Together they are responsible for the high level of peptidoglycan -deacetylation in and the consequent resistance to lysozyme. We also characterized a third enzyme, PgdB, as a glucosamine -deacetylase. However, its impact on -deacetylation and lysozyme resistance is limited, and its physiological role remains to be dissected. Finally, given the influence of peptidoglycan -deacetylation on host defense against pathogens, we investigated the virulence and colonization ability of the mutants. Unlike what has been shown in other pathogenic bacteria, a lack of -deacetylation in is not linked to a decrease in virulence.
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http://dx.doi.org/10.1074/jbc.RA119.012442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864072PMC
December 2020

The cryo-electron microscopy supramolecular structure of the bacterial stressosome unveils its mechanism of activation.

Nat Commun 2019 07 8;10(1):3005. Epub 2019 Jul 8.

Département de Biologie Cellulaire et Infection, Institut Pasteur, Unité des Interactions Bactéries-Cellules, 75015, Paris, France.

How the stressosome, the epicenter of the stress response in bacteria, transmits stress signals from the environment has remained elusive. The stressosome consists of multiple copies of three proteins RsbR, RsbS and RsbT, a kinase that is important for its activation. Using cryo-electron microscopy, we determined the atomic organization of the Listeria monocytogenes stressosome at 3.38 Å resolution. RsbR and RsbS are organized in a 60-protomers truncated icosahedron. A key phosphorylation site on RsbR (T209) is partially hidden by an RsbR flexible loop, whose "open" or "closed" position could modulate stressosome activity. Interaction between three glutamic acids in the N-terminal domain of RsbR and the membrane-bound mini-protein Prli42 is essential for Listeria survival to stress. Together, our data provide the atomic model of the stressosome core and highlight a loop important for stressosome activation, paving the way towards elucidating the mechanism of signal transduction by the stressosome in bacteria.
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http://dx.doi.org/10.1038/s41467-019-10782-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614362PMC
July 2019

Quantification of low abundance Yersinia pestis markers in dried blood spots by immuno-capture and quantitative high-resolution targeted mass spectrometry.

Eur J Mass Spectrom (Chichester) 2019 Jun;25(3):268-277

1 Service de Pharmacologie et d'Immunoanalyse (SPI), CEA, INRA, Université Paris-Saclay, Gif sur Yvette, France.

Plague, caused by the bacterium Yersinia pestis, is still present in several countries worldwide. Besides, Y. pestis has been designated as Tier 1 agent, the highest rank of bioterrorism agents. In this context, reliable diagnostic methods are of great importance. Here, we have developed an original workflow based upon dried blood spot for simplified sampling of clinical specimens, and specific immuno-mass spectrometry monitoring of Y. pestis biomarkers. Targeted proteins were selectively enriched from dried blood spot extracts by multiplex immunocapture using antibody-coated magnetic beads. After accelerated on-beads digestion, proteotypic peptides were monitored by multiplex LC-MS/MS through the parallel reaction monitoring mode. The DBS-IC-MS assay was designed to quantify both F1 and LcrV antigens, although 10-fold lower sensitivity was observed with LcrV. The assay was successfully validated for F1 with a lower limit of quantification at 5 ng·mL in spiked blood, corresponding to only 0.1 ng on spots. In vivo quantification of F1 in blood and organ samples was demonstrated in the mouse model of pneumonic plague. The new assay could help to simplify the laboratory confirmation of positive point of care F1 dipstick.
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http://dx.doi.org/10.1177/1469066718795978DOI Listing
June 2019

-Deacetylases required for muramic-δ-lactam production are involved in sporulation, germination, and heat resistance.

J Biol Chem 2018 11 28;293(47):18040-18054. Epub 2018 Sep 28.

From the EA4043 Unité Bactéries Pathogènes et Santé (UBaPS), Université Paris-Sud, Université Paris-Saclay, 92290 Châtenay-Malabry,. Electronic address:

Spores are produced by many organisms as a survival mechanism activated in response to several environmental stresses. Bacterial spores are multilayered structures, one of which is a peptidoglycan layer called the cortex, containing muramic-δ-lactams that are synthesized by at least two bacterial enzymes, the muramoyl-l-alanine amidase CwlD and the -deacetylase PdaA. This study focused on the spore cortex of , a Gram-positive, toxin-producing anaerobic bacterial pathogen that can colonize the human intestinal tract and is a leading cause of antibiotic-associated diarrhea. Using ultra-HPLC coupled with high-resolution MS, here we found that the spore cortex of the 630Δ strain differs from that of Among these differences, the muramic-δ-lactams represented only 24% in , compared with 50% in CD630_14300 and CD630_27190 were identified as genes encoding the -deacetylases PdaA1 and PdaA2, required for muramic-δ-lactam synthesis. In a mutant, only 0.4% of all muropeptides carried a muramic-δ-lactam modification, and muramic-δ-lactams were absent in the cortex of a double mutant. Of note, the mutant exhibited decreased sporulation, altered germination, decreased heat resistance, and delayed virulence in a hamster infection model. These results suggest a much greater role for muramic-δ-lactams in than in other bacteria, including In summary, the spore cortex of contains lower levels of muramic-δ-lactams than that of , and PdaA1 is the major -deacetylase for muramic-δ-lactam biosynthesis in , contributing to sporulation, heat resistance, and virulence.
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http://dx.doi.org/10.1074/jbc.RA118.004273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6254358PMC
November 2018

Biochemical and biophysical combined study of bicarinalin, an ant venom antimicrobial peptide.

Peptides 2016 05 4;79:103-13. Epub 2016 Apr 4.

Equipe EA 4357, VAcBio, Université de Toulouse, CUFR Champollion, Place de Verdun, 81012 Albi, France. Electronic address:

We have recently characterized bicarinalin as the most abundant peptide from the venom of the ant Tetramorium bicarinatum. This antimicrobial peptide is active against Staphylococcus and Enterobacteriaceae. To further investigate the antimicrobial properties of this cationic and cysteine-free peptide, we have studied its antibacterial, antifungal and antiparasitic activities on a large array of microorganisms. Bicarinalin was active against fifteen microorganisms with minimal inhibitory concentrations ranging from 2 and 25μmolL(-1). Cronobacter sakazakii, Salmonella enterica, Candida albicans, Aspergilus niger and Saccharomyces cerevisiae were particularly susceptible to this novel antimicrobial peptide. Resistant strains of Staphylococcus aureus, Pseudomonas aeruginosa and C. albicans were as susceptible as the canonical strains. Interestingly, bicarinalin was also active against the parasite Leishmania infantum with a minimal inhibitory concentrations of 2μmolL(-1). The bicarinalin pre-propeptide cDNA sequence has been determined using a combination of degenerated primers with RACE PCR strategy. Interestingly, the N-terminal domain of bicarinalin pre-propeptide exhibited sequence similarity with the pilosulin antimicrobial peptide family previously described in the Myrmecia venoms. Moreover, using SYTOX green uptake assay, we showed that, for all the tested microorganisms, bicarinalin acted through a membrane permeabilization mechanism. Two dimensional-NMR experiments showed that bicarinalin displayed a 10 residue-long α-helical structure flanked by two N- and C-terminal disordered regions. This partially amphipathic helix may explain the membrane permeabilization mechanism of bicarinalin observed in this study. Finally, therapeutic value of bicarinalin was highlighted by its low cytotoxicity against human lymphocytes at bactericidal concentrations and its long half-life in human serum which was around 15h.
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http://dx.doi.org/10.1016/j.peptides.2016.04.001DOI Listing
May 2016

Identification and characterization of a novel antimicrobial peptide from the venom of the ant Tetramorium bicarinatum.

Peptides 2012 Dec 7;38(2):363-70. Epub 2012 Sep 7.

Equipe VacBio EA 4357, PRES Université Toulouse, CUFR JF Champollion, Place de Verdun, 81012 Albi, France.

A novel antimicrobial peptide, named Bicarinalin, has been isolated from the venom of the ant Tetramorium bicarinatum. Its amino acid sequence has been determined by de novo sequencing using mass spectrometry and by Edman degradation. Bicarinalin contained 20 amino acid residues and was C-terminally amidated as the majority of antimicrobial peptides isolated to date from insect venoms. Interestingly, this peptide had a linear structure and exhibited no meaningful similarity with any known peptides. Antibacterial activities against Staphylococcus aureus and S. xylosus strains were evaluated using a synthetic replicate. Bicarinalin had a potent and broad antibacterial activity of the same magnitude as Melittin and other hymenopteran antimicrobial peptides such as Pilosulin or Defensin. Moreover, this antimicrobial peptide has a weak hemolytic activity compared to Melittin on erythrocytes, suggesting potential for development into an anti-infective agent for use against emerging antibiotic-resistant pathogens.
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http://dx.doi.org/10.1016/j.peptides.2012.08.018DOI Listing
December 2012

Paralyzing action from a distance in an arboreal African ant species.

PLoS One 2011 14;6(12):e28571. Epub 2011 Dec 14.

Venoms and Biological Activities Laboratory, EA 4357, PRES-Université de Toulouse, Jean-François Champollion University Center, Albi, France.

Due to their prowess in interspecific competition and ability to catch a wide range of arthropod prey (mostly termites with which they are engaged in an evolutionary arms race), ants are recognized as a good model for studying the chemicals involved in defensive and predatory behaviors. Ants' wide diversity of nesting habits and relationships with plants and prey types implies that these chemicals are also very diverse. Using the African myrmicine ant Crematogaster striatula as our focal species, we adopted a three-pronged research approach. We studied the aggressive and predatory behaviors of the ant workers, conducted bioassays on the effect of their Dufour gland contents on termites, and analyzed these contents. (1) The workers defend themselves or eliminate termites by orienting their abdominal tip toward the opponent, stinger protruded. The chemicals emitted, apparently volatile, trigger the recruitment of nestmates situated in the vicinity and act without the stinger having to come into direct contact with the opponent. Whereas alien ants competing with C. striatula for sugary food sources are repelled by this behavior and retreat further and further away, termites defend their nest whatever the danger. They face down C. striatula workers and end up by rolling onto their backs, their legs batting the air. (2) The bioassays showed that the toxicity of the Dufour gland contents acts in a time-dependent manner, leading to the irreversible paralysis, and, ultimately, death of the termites. (3) Gas chromatography-mass spectrometry analyses showed that the Dufour gland contains a mixture of mono- or polyunsaturated long-chain derivatives, bearing functional groups like oxo-alcohols or oxo-acetates. Electrospray ionization-mass spectrometry showed the presence of a molecule of 1584 Da that might be a large, acetylated alkaloid capable of splitting into smaller molecules that could be responsible for the final degree of venom toxicity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0028571PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3237465PMC
August 2012