Personne :
Villion, Manuela

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Département de biochimie, microbiologie et bio-informatique, Faculté des sciences et de génie, Université Laval
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Voici les éléments 1 - 10 sur 13
  • Publication
    Accès libre
    Phages hijack a host's defence
    (Nature Publishing Group, 2013-02-27) Moineau, Sylvain; Villion, Manuela
    The discovery that some viruses use a defence mechanism known as a CRISPR/Cas system beautifully illustrates the evolutionary tit-for-tat between viruses and the bacteria they infect.
  • Publication
    Accès libre
    CRISPR-Cas and restriction–modification systems are compatible and increase phage resistance
    (Nature Publishing Group, 2013-07-02) Moineau, Sylvain; Dupuis, Marie-Ève; Magadán, Alfonso H.; Villion, Manuela
    Bacteria have developed a set of barriers to protect themselves against invaders such as phage and plasmid nucleic acids. Different prokaryotic defence systems exist and at least two of them directly target the incoming DNA: restriction–modification (R-M) and CRISPR-Cas systems. On their own, they are imperfect barriers to invasion by foreign DNA. Here, we show that R-M and CRISPR-Cas systems are compatible and act together to increase the overall phage resistance of a bacterial cell by cleaving their respective target sites. Furthermore, we show that the specific methylation of phage DNA does not impair CRISPR-Cas acquisition or interference activities. Taken altogether, both mechanisms can be leveraged to decrease phage contaminations in processes relying on bacterial growth and/or fermentation.
  • Publication
    Inactivation of dairy bacteriophages by commercial sanitizers and disinfectants
    (Elsevier, 2013-11-19) Duchaine, Caroline; Moineau, Sylvain; Labrie, Simon; Campagna, Céline; Villion, Manuela
    Many commercial sanitizers and disinfectants have been used over the years to control microbial contamination but their efficacy on phages is often unknown. Here, 23 commercial chemical products, including 21 food-grade sanitizers were tested against virulent dairy phages. These food-grade chemicals included oxidizing agents, halogenated agents, alcohols, quaternary ammonium compounds, anionic acids, iodine-based acids, and an amphoteric chemical. Phage P008 was first exposed to each sanitizer for 2 and 15 min at room temperature and at two different concentrations, namely the lowest and highest no-rinse sanitizing concentrations. Organic matter (whey or milk) was also added to the testing solutions. At the end of the exposure period, the test solution was neutralized and the number of infectious phages was determined by plaque assays. The five most efficient sanitizers against phage P008 (< 4 log of inactivation) were then tested against virulent lactococcal phages P008, CB13, AF6, P1532 of the 936 group, P001 (c2), Q54, and 1358 as well as Lactobacillus plantarum phage B1 and Streptococcus thermophilus phage 2972 using the same protocol. The oxidizing agents and the quaternary ammonium compounds were the most efficient against all phages although phages CB13 and P1532 were less sensitive to these chemicals than the other phages. This study may help in the selection of appropriate chemicals for controlling phage contamination in industrial factories and research laboratories.
  • Publication
    Involvement of the major capsid protein and two early-expressed phage genes in the activity of the lactococcal abortive infection mechanism abiT
    (American Society for Microbiology, 2012-09-07) Tremblay, Denise; Moisan, Maxim; Moineau, Sylvain; Labrie, Simon; Magadán, Alfonso H.; Campanacci, Valérie; Villion, Manuela; Cambillau, Christian
    The dairy industry uses the mesophilic, Gram-positive, lactic acid bacterium (LAB) Lactococcus lactis to produce an array of fermented milk products. Milk fermentation processes are susceptible to contamination by virulent phages, but a plethora of phage control strategies are available. One of the most efficient is to use LAB strains carrying phage resistance systems such as abortive infection (Abi) mechanisms. Yet, the mode of action of most Abi systems remains poorly documented. Here, we shed further light on the antiviral activity of the lactococcal AbiT system. Twenty-eight AbiT-resistant phage mutants derived from the wild-type AbiT-sensitive lactococcal phages p2, bIL170, and P008 were isolated and characterized. Comparative genomic analyses identified three different genes that were mutated in these virulent AbiT-insensitive phage derivatives: e14 (bIL170 [e14bIL170]), orf41 (P008 [orf41P008]), and orf6 (p2 [orf6p2] and P008 [orf6P008]). The genes e14bIL170 and orf41P008 are part of the early-expressed genomic region, but bioinformatic analyses did not identify their putative function. orf6 is found in the phage morphogenesis module. Antibodies were raised against purified recombinant ORF6, and immunoelectron microscopy revealed that it is the major capsid protein (MCP). Coexpression in L. lactis of ORF6p2 and ORF5p2, a protease, led to the formation of procapsids. To our knowledge, AbiT is the first Abi system involving distinct phage genes.
  • Publication
    Accès libre
    Bacteriophages of Lactobacillus
    (Frontiers in Bioscience Publications, 2009-01-01) Moineau, Sylvain; Villion, Manuela
    In this review, we are listing Lactobacillus phages that have been reported in peer-reviewed articles published since 1960. Putative phages that are defective or have not been shown to be infectious, such as phage-like particles, are not discussed. Our literature searches led to the identification of 231 Lactobacillus phages, 186 of which have been observed by electron microscopy, with 109 belonging to the Siphoviridae family, 76 to the Myoviridae family, and 1 to the Podoviridae family. Model phages infecting Lb delbrueckii, casei, rhamnosus, plantarum, and gasseri are highlighted, as well as prophages of Lactobacillus hosts. To date, nine complete Lactobacillus phage genomes are available for comparisons and evolution studies. Features such as phage receptors and endolysins are also reviewed, as well as phage-derived genetic tools. Lactobacillus phage research has progressed significantly over the past decade but a thorough understanding of their biology is still lacking. Because of the risks they represent and the knowledge gaps that need to be filled, the outlook for research on Lactobacillus phages is bright.
  • Publication
    Accès libre
    The double-edged sword of CRISPR-Cas systems
    (Nature Publishing Group, 2012-09-04) Moineau, Sylvain; Villion, Manuela
    A recent paper gives the details on how specific small RNAs can program a protein to cleave an undesired piece of DNA and to provide immunity to a microbial cell.
  • Publication
    Accès libre
    The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA
    (Macmillan, 2010-11-03) Garneau, Josiane; Devillers, Rodolphe; Moineau, Sylvain; Romero, Dennis A.; Dupuis, Marie-Ève; Magadán, Alfonso H.; Boyaval, Patrick; Villion, Manuela; Fremaux, Christophe; Horvath, Philippe
    Bacteria and Archaea have developed several defence strategies against foreign nucleic acids such as viral genomes and plasmids. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) loci together with cas (CRISPR-associated) genes form the CRISPR/Cas immune system, which involves partially palindromic repeats separated by short stretches of DNA called spacers, acquired from extrachromosomal elements. It was recently demonstrated that these variable loci can incorporate spacers from infecting bacteriophages and then provide immunity against subsequent bacteriophage infections in a sequence-specific manner. Here we show that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading to plasmid loss. Acquired spacers that match antibiotic-resistance genes provide a novel means to naturally select bacteria that cannot uptake and disseminate such genes. We also provide in vivo evidence that the CRISPR1/Cas system specifically cleaves plasmid and bacteriophage double-stranded DNA within the proto-spacer, at specific sites. Our data show that the CRISPR/Cas immune system is remarkably adapted to cleave invading DNA rapidly and has the potential for exploitation to generate safer microbial strains.
  • Publication
    Accès libre
    Cleavage of phage DNA by the Streptococcus thermophilus CRISPR3-Cas system
    (Public Library of Science, 2012-07-20) Moineau, Sylvain; Dupuis, Marie-Ève; Magadán, Alfonso H.; Villion, Manuela
    Streptococcus thermophilus, similar to other Bacteria and Archaea, has developed defense mechanisms to protect cells against invasion by foreign nucleic acids, such as virus infections and plasmid transformations. One defense system recently described in these organisms is the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats loci coupled to CRISPR-associated genes). Two S. thermophilus CRISPR-Cas systems, CRISPR1-Cas and CRISPR3-Cas, have been shown to actively block phage infection. The CRISPR1-Cas system interferes by cleaving foreign dsDNA entering the cell in a length-specific and orientation-dependant manner. Here, we show that the S. thermophilus CRISPR3-Cas system acts by cleaving phage dsDNA genomes at the same specific position inside the targeted protospacer as observed with the CRISPR1-Cas system. Only one cleavage site was observed in all tested strains. Moreover, we observed that the CRISPR1-Cas and CRISPR3-Cas systems are compatible and, when both systems are present within the same cell, provide increased resistance against phage infection by both cleaving the invading dsDNA. We also determined that overall phage resistance efficiency is correlated to the total number of newly acquired spacers in both CRISPR loci.
  • Publication
    CRISPR/Cas, un système immunitaire bactérien
    (Lavoisier, 2011-06-01) Moineau, Sylvain; Villion, Manuela
    Les bactéries vivent dans des environnements en constante évolution où elles subissent régulièrement les assauts d’éléments étrangers, tels des phages et des plasmides, qui tentent de s’introduire dans leur enceinte cellulaire. Aussi ont-elles développé une panoplie de systèmes de défense. Le dernier découvert, le système CRISPR/Cas, suscite un intérêt considérable dans divers champs d’activité académiques et industriels.
  • Publication
    Accès libre
    Crystal structure of ORF12 from Lactococcus lactis phage p2 identifies a tape measure protein chaperone
    (American Society for Microbiology, 2009-01-16) Siponen, Marina; Moineau, Sylvain; Sciara, Giuliano; Villion, Manuela; Spinelli, Silvia; Lichière, Julie; Cambillau, Christian; Campanacci, Valérie
    We report here the characterization of the nonstructural protein ORF12 of the virulent lactococcal phage p2, which belongs to the Siphoviridae family. ORF12 was produced as a soluble protein, which forms large oligomers (6- to 15-mers) in solution. Using anti-ORF12 antibodies, we have confirmed that ORF12 is not found in the virion structure but is detected in the second half of the lytic cycle, indicating that it is a late-expressed protein. The structure of ORF12, solved by single anomalous diffraction and refined at 2.9-Å resolution, revealed a previously unknown fold as well as the presence of a hydrophobic patch at its surface. Furthermore, crystal packing of ORF12 formed long spirals in which a hydrophobic, continuous crevice was identified. This crevice exhibited a repeated motif of aromatic residues, which coincided with the same repeated motif usually found in tape measure protein (TMP), predicted to form helices. A model of a complex between ORF12 and a repeated motif of the TMP of phage p2 (ORF14) was generated, in which the TMP helix fitted exquisitely in the crevice and the aromatic patches of ORF12. We suggest, therefore, that ORF12 might act as a chaperone for TMP hydrophobic repeats, maintaining TMP in solution during the tail assembly of the lactococcal siphophage p2.