Publications by authors named "Sylviane Furlan"

11 Publications

  • Page 1 of 1

Complete Structure of the Enterococcal Polysaccharide Antigen (EPA) of Vancomycin-Resistant Enterococcus faecalis V583 Reveals that EPA Decorations Are Teichoic Acids Covalently Linked to a Rhamnopolysaccharide Backbone.

mBio 2020 04 28;11(2). Epub 2020 Apr 28.

Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, Jouy-en-Josas, France

All enterococci produce a complex polysaccharide called the nterococcal olysaccharide ntigen (EPA). This polymer is required for normal cell growth and division and for resistance to cephalosporins and plays a critical role in host-pathogen interaction. The EPA contributes to host colonization and is essential for virulence, conferring resistance to phagocytosis during the infection. Recent studies revealed that the "decorations" of the EPA polymer, encoded by genetic loci that are variable between isolates, underpin the biological activity of this surface polysaccharide. In this work, we investigated the structure of the EPA polymer produced by the high-risk enterococcal clonal complex V583. We analyzed purified EPA from the wild-type strain and a mutant lacking decorations and elucidated the structure of the EPA backbone and decorations. We showed that the rhamnan backbone of EPA is composed of a hexasaccharide repeat unit of C2- and C3-linked rhamnan chains, partially substituted in the C3 position by α-glucose (α-Glc) and in the C2 position by β--acetylglucosamine (β-GlcNAc). The so-called "EPA decorations" consist of phosphopolysaccharide chains corresponding to teichoic acids covalently bound to the rhamnan backbone. The elucidation of the complete EPA structure allowed us to propose a biosynthetic pathway, a first essential step toward the design of antimicrobials targeting the synthesis of this virulence factor. Enterococci are opportunistic pathogens responsible for hospital- and community-acquired infections. All enterococci produce a surface polysaccharide called EPA (nterococcal olysaccharide ntigen) required for biofilm formation, antibiotic resistance, and pathogenesis. Despite the critical role of EPA in cell growth and division and as a major virulence factor, no information is available on its structure. Here, we report the complete structure of the EPA polymer produced by the model strain V583. We describe the structure of the EPA backbone, made of a rhamnan hexasaccharide substituted by Glc and GlcNAc residues, and show that teichoic acids are covalently bound to this rhamnan chain, forming the so-called "EPA decorations" essential for host colonization and pathogenesis. This report represents a key step in efforts to identify the structural properties of EPA that are essential for its biological activity and to identify novel targets to develop preventive and therapeutic approaches against enterococci.
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http://dx.doi.org/10.1128/mBio.00277-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7188991PMC
April 2020

Fitness Restoration of a Genetically Tractable Enterococcus faecalis V583 Derivative To Study Decoration-Related Phenotypes of the Enterococcal Polysaccharide Antigen.

mSphere 2019 07 10;4(4). Epub 2019 Jul 10.

UMR Micalis, INRA, AgroParisTech, Université Paris-Saclay, UMR1319, Jouy-en-Josas, France

Commensal and generally harmless in healthy individuals, causes opportunistic infections in immunocompromised patients. Plasmid-cured strain VE14089, derived from sequenced reference strain V583, is widely used for functional studies due to its improved genetic amenability. Although strain VE14089 has no major DNA rearrangements, with the exception of an ∼20-kb integrated region of pTEF1 plasmid, the strain presented significant growth differences from the V583 reference strain of our collection (renamed VE14002). In the present study, genome sequencing of strain VE14089 identified additional point mutations. Excision of the integrated pTEF1 plasmid region and sequential restoration of wild-type alleles showing nonsilent mutations were performed to obtain the VE18379 reference-derivative strain. Recovery of the growth ability of the restored VE18379 strain at a level similar to that seen with the reference strain points to GreA and Spx as bacterial fitness determinants. Virulence potential in and intestinal colonization in mouse demonstrated host adaptation of the VE18379 strain equivalent to VE14002 host adaptation. We further demonstrated that deletion of the 16.8-kb variable region of the locus recapitulates the key role of Epa decoration in host adaptation, providing a genetic system to study the role of specific -variable regions in host adaptation independently of other genetic variations. strain VE14089 was derived from V583 cured of its plasmids. Although VE14089 had no major DNA rearrangements, it presented significant growth and host adaptation differences from the reference strain V583 of our collection. To construct a strain with better fitness, we sequenced the genome of VE14089, identified single nucleotide polymorphisms (SNPs), and repaired the genes that could account for these changes. Using this reference-derivative strain, we provide a novel genetic system to understand the role of the variable region of in the enterococcal lifestyle.
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http://dx.doi.org/10.1128/mSphere.00310-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6620374PMC
July 2019

Countermeasures Defeat a Virulent Bacteriophage.

Viruses 2019 01 10;11(1). Epub 2019 Jan 10.

Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France.

is an opportunistic pathogen that has emerged as a major cause of nosocomial infections worldwide. Many clinical strains are indeed resistant to last resort antibiotics and there is consequently a reawakening of interest in exploiting virulent phages to combat them. However, little is still known about phage receptors and phage resistance mechanisms in enterococci. We made use of a prophageless derivative of the well-known clinical strain V583 to isolate a virulent phage belonging to the subfamily and to the P68 genus that we named Idefix. Interestingly, most isolates of tested-including V583-were resistant to this phage and we investigated more deeply into phage resistance mechanisms. We found that V583 prophage 6 was particularly efficient in resisting Idefix infection thanks to a new abortive infection (Abi) mechanism, which we designated Abiα. It corresponded to the Pfam domain family with unknown function DUF4393 and conferred a typical Abi phenotype by causing a premature lysis of infected . The gene is widespread among prophages of enterococci and other Gram-positive bacteria. Furthermore, we identified two genes involved in the synthesis of the side chains of the surface rhamnopolysaccharide that are important for Idefix adsorption. Interestingly, mutants in these genes arose at a frequency of ~10 resistant mutants per generation, conferring a supplemental bacterial line of defense against Idefix.
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http://dx.doi.org/10.3390/v11010048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356687PMC
January 2019

Another Brick in the Wall: a Rhamnan Polysaccharide Trapped inside Peptidoglycan of .

mBio 2017 09 12;8(5). Epub 2017 Sep 12.

Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France

Polysaccharides are ubiquitous components of the Gram-positive bacterial cell wall. In , a polysaccharide pellicle (PSP) forms a layer at the cell surface. The PSP structure varies among lactococcal strains; in MG1363, the PSP is composed of repeating hexasaccharide phosphate units. Here, we report the presence of an additional neutral polysaccharide in MG1363 that is a rhamnan composed of α-l-Rha trisaccharide repeating units. This rhamnan is still present in mutants devoid of the PSP, indicating that its synthesis can occur independently of PSP synthesis. High-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) analysis of whole bacterial cells identified a PSP at the surface of wild-type cells. In contrast, rhamnan was detected only at the surface of PSP-negative mutant cells, indicating that rhamnan is located underneath the surface-exposed PSP and is trapped inside peptidoglycan. The genetic determinants of rhamnan biosynthesis appear to be within the same genetic locus that encodes the PSP biosynthetic machinery, except the gene encoding the initiating glycosyltransferase. We present a model of rhamnan biosynthesis based on an ABC transporter-dependent pathway. Conditional mutants producing reduced amounts of rhamnan exhibit strong morphological defects and impaired division, indicating that rhamnan is essential for normal growth and division. Finally, a mutation leading to reduced expression of , encoding a protein of the LytR-CpsA-Psr (LCP) family, was shown to severely affect cell wall structure. In mutant cells, in contrast to wild-type cells, rhamnan was detected by HR-MAS NMR, suggesting that LcpA participates in the attachment of rhamnan to peptidoglycan. In the cell wall of Gram-positive bacteria, the peptidoglycan sacculus is considered the major structural component, maintaining cell shape and integrity. It is decorated with other glycopolymers, including polysaccharides, the roles of which are not fully elucidated. In the ovococcus , a polysaccharide with a different structure between strains forms a layer at the bacterial surface and acts as the receptor for various bacteriophages that typically exhibit a narrow host range. The present report describes the identification of a novel polysaccharide in the cell wall, a rhamnan that is trapped inside the peptidoglycan and covalently bound to it. We propose a model of rhamnan synthesis based on an ABC transporter-dependent pathway. Rhamnan appears as a conserved component of the lactococcal cell wall playing an essential role in growth and division, thus highlighting the importance of polysaccharides in the cell wall integrity of Gram-positive ovococci.
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http://dx.doi.org/10.1128/mBio.01303-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5596347PMC
September 2017

Regulation of Cell Wall Plasticity by Nucleotide Metabolism in Lactococcus lactis.

J Biol Chem 2016 May 28;291(21):11323-36. Epub 2016 Mar 28.

Micalis Institute and

To ensure optimal cell growth and separation and to adapt to environmental parameters, bacteria have to maintain a balance between cell wall (CW) rigidity and flexibility. This can be achieved by a concerted action of peptidoglycan (PG) hydrolases and PG-synthesizing/modifying enzymes. In a search for new regulatory mechanisms responsible for the maintenance of this equilibrium in Lactococcus lactis, we isolated mutants that are resistant to the PG hydrolase lysozyme. We found that 14% of the causative mutations were mapped in the guaA gene, the product of which is involved in purine metabolism. Genetic and transcriptional analyses combined with PG structure determination of the guaA mutant enabled us to reveal the pivotal role of the pyrB gene in the regulation of CW rigidity. Our results indicate that conversion of l-aspartate (l-Asp) to N-carbamoyl-l-aspartate by PyrB may reduce the amount of l-Asp available for PG synthesis and thus cause the appearance of Asp/Asn-less stem peptides in PG. Such stem peptides do not form PG cross-bridges, resulting in a decrease in PG cross-linking and, consequently, reduced PG thickness and rigidity. We hypothesize that the concurrent utilization of l-Asp for pyrimidine and PG synthesis may be part of the regulatory scheme, ensuring CW flexibility during exponential growth and rigidity in stationary phase. The fact that l-Asp availability is dependent on nucleotide metabolism, which is tightly regulated in accordance with the growth rate, provides L. lactis cells the means to ensure optimal CW plasticity without the need to control the expression of PG synthesis genes.
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http://dx.doi.org/10.1074/jbc.M116.714303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4900277PMC
May 2016

Surface proteome analysis of a natural isolate of Lactococcus lactis reveals the presence of pili able to bind human intestinal epithelial cells.

Mol Cell Proteomics 2013 Dec 3;12(12):3935-47. Epub 2013 Sep 3.

INRA, UMR1319 Micalis, F-78350 Jouy-en-Josas, France;

Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells.
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http://dx.doi.org/10.1074/mcp.M113.029066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3861735PMC
December 2013

Cell surface of Lactococcus lactis is covered by a protective polysaccharide pellicle.

J Biol Chem 2010 Apr 27;285(14):10464-71. Epub 2010 Jan 27.

INRA, UMR1319 Micalis, Domaine de Vilvert, F-78352 Jouy-en-Josas, France.

In Gram-positive bacteria, the functional role of surface polysaccharides (PS) that are not of capsular nature remains poorly understood. Here, we report the presence of a novel cell wall PS pellicle on the surface of Lactococcus lactis. Spontaneous PS-negative mutants were selected using semi-liquid growth conditions, and all mutations were mapped in a single chromosomal locus coding for PS biosynthesis. PS molecules were shown to be composed of hexasaccharide phosphate repeating units that are distinct from other bacterial PS. Using complementary atomic force and transmission electron microscopy techniques, we showed that the PS layer forms an outer pellicle surrounding the cell. Notably, we found that this cell wall layer confers a protective barrier against host phagocytosis by murine macrophages. Altogether, our results suggest that the PS pellicle could represent a new cell envelope structural component of Gram-positive bacteria.
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http://dx.doi.org/10.1074/jbc.M109.082958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2856253PMC
April 2010

SpxB regulates O-acetylation-dependent resistance of Lactococcus lactis peptidoglycan to hydrolysis.

J Biol Chem 2007 Jul 7;282(27):19342-54. Epub 2007 May 7.

Unité Bactéries Lactiques et Pathogènes Opportunistes, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex, France.

Endogenous peptidoglycan (PG)-hydrolyzing enzymes, the autolysins, are needed to relax the rigid PG sacculus to allow bacterial cell growth and separation. PGs of pathogens and commensal bacteria may also be degraded by hydrolases of animal origin (lysozymes), which act as antimicrobials. The genetic mechanisms regulating PG resistance to hydrolytic degradation were dissected in the Gram-positive bacterium Lactococcus lactis. We found that the ability of L. lactis to counteract PG hydrolysis depends on the degree of acetylation. Overexpression of PG O-acetylase (encoded by oatA) led to bacterial growth arrest, indicating the potential lethality of oatA and a need for its tight regulation. A novel regulatory factor, SpxB (previously denoted as YneH), exerted a positive effect on oatA expression. Our results indicate that SpxB binding to RNA polymerase constitutes a previously missing link in the multistep response to cell envelope stress, provoked by PG hydrolysis with lysozyme. We suggest that the two-component system CesSR responds to this stress by inducing SpxB, thus favoring its interactions with RNA polymerase. Induction of PG O-acetylation by this cascade renders it resistant to hydrolysis.
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http://dx.doi.org/10.1074/jbc.M611308200DOI Listing
July 2007

Identification of an essential gene responsible for D-Asp incorporation in the Lactococcus lactis peptidoglycan crossbridge.

Mol Microbiol 2006 Dec;62(6):1713-24

Unité Bactéries Lactiques et Pathogènes Opportunistes, INRA, 78352 Jouy-en-Josas Cedex France.

Bacteria such as Lactococcus lactis have D-aspartate (D-Asp) or its amidated derivative D-asparagine (D-Asn), in their peptidoglycan (PG) interpeptide crossbridge. We performed a subtractive genome analysis to identify L. lactis gene yxbA, orthologues of which being present only in bacteria containing D-amino acids in their PG crossbridge, but absent from those that instead insert L-amino acids or glycine. Inactivation of yxbA required a complementing Streptococcus pneumoniae murMN genes, which express enzymes that incorporate L-Ser-L-Ala or L-Ala-L-Ala in the PG crossbridge. Our results show that (i) yxbA encodes D-Asp ligase responsible for incorporation of D-Asp in the PG crossbridge, and we therefore renamed it as aslA, (ii) it is an essential gene, which makes its product a potential target for specific antimicrobials, (iii) the absence of D-Asp may be complemented by L-Ser-L-Ala or L-Ala-L-Ala in the L. lactis PG, indicating that the PG synthesis machinery is not selective for the side-chain residues, and (iv) lactococcal strains having L-amino acids in their PG crossbridge display defects in cell wall integrity, but are able to efficiently anchor cell wall proteins, indicating relative flexibility of lactococcal transpeptidation reactions with respect to changes in PG sidechain composition.
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http://dx.doi.org/10.1111/j.1365-2958.2006.05474.xDOI Listing
December 2006

C-terminal WxL domain mediates cell wall binding in Enterococcus faecalis and other gram-positive bacteria.

J Bacteriol 2007 Feb 8;189(4):1244-53. Epub 2006 Sep 8.

Unité des Bactéries Lactiques et Pathogènes Opportunistes, INRA, Jouy-en-Josas, France.

Analysis of the genome sequence of Enterococcus faecalis clinical isolate V583 revealed novel genes encoding surface proteins. Twenty-seven of these proteins, annotated as having unknown functions, possess a putative N-terminal signal peptide and a conserved C-terminal region characterized by a novel conserved domain designated WxL. Proteins having similar characteristics were also detected in other low-G+C-content gram-positive bacteria. We hypothesized that the WxL region might be a determinant of bacterial cell location. This hypothesis was tested by generating protein fusions between the C-terminal regions of two WxL proteins in E. faecalis and a nuclease reporter protein. We demonstrated that the C-terminal regions of both proteins conferred a cell surface localization to the reporter fusions in E. faecalis. This localization was eliminated by introducing specific deletions into the domains. Interestingly, exogenously added protein fusions displayed binding to whole cells of various gram-positive bacteria. We also showed that the peptidoglycan was a binding ligand for WxL domain attachment to the cell surface and that neither proteins nor carbohydrates were necessary for binding. Based on our findings, we propose that the WxL region is a novel cell wall binding domain in E. faecalis and other gram-positive bacteria.
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http://dx.doi.org/10.1128/JB.00773-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1797349PMC
February 2007

Comparison of the thickening properties of four Lactobacillus delbrueckii subsp. bulgaricus strains and physicochemical characterization of their exopolysaccharides.

FEMS Microbiol Lett 2003 Apr;221(2):285-91

Unité de Recherches Laitières et Génétique Appliquée, INRA, Jouy-en-Josas, France.

It is now well established that physicochemical properties of exopolysaccharides (EPS) can vary between strains of a given species and according to growth conditions. The EPS production of four strains of Lactobacillus bulgaricus was monitored during growth in milk and in a chemically defined media. All strains, including the non-ropy one, produced EPS. The monosaccharide composition, molar mass (M(w)), and intrinsic viscosity of these EPS were determined and compared. Further characterization using high-performance size-exclusion chromatography revealed the presence of two fractions in all EPS: one fraction exhibited a high M(w) and a high intrinsic viscosity while the other had a low M(w) and a low intrinsic viscosity. Strikingly, the EPS synthesized by the non-ropy strain was mainly composed of the low-M(w) fraction while for the ropy strains, the fraction of high M(w) varied between 43 and 90%. According to our results, we propose that the ratio between the high-M(w) and low-M(w) fractions is critical for the texturing properties of L. bulgaricus EPS.
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http://dx.doi.org/10.1016/S0378-1097(03)00214-3DOI Listing
April 2003
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