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Moineau, Sylvain

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Moineau

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Sylvain

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Université Laval. Département de biochimie, de microbiologie et de bio-informatique

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  • PublicationRestreint
    Comparison of polycarbonate and polytetrafluoroethylene filters for sampling of airborne bacteriophages
    (Elsevier, 2010-01-22) Duchaine, Caroline; Gendron, Louis; Moineau, Sylvain; Massé, Daniel; Verreault, Daniel; Rousseau, Geneviève M.
    Aerosolized coliphage phiX174 and lactococcal phage P008 were sampled with two types of filter, polycarbonate (PC) and polytetrafluoroethylene (PTFE). The recovery was determined by plaque assays and quantitative polymerase chain reaction (qPCR). Recovery by qPCR was greater than by culture and PC filters outperformed PTFE filters both by culture and by qPCR relative recovery. The results of the plaque assays showed that the infectivity of the recovered phages was affected by the aerosolization/air sampling. The presence of viruses in air samples should be determined by culture-independent assays.
  • PublicationRestreint
    Lactococcus lactis type III-A CRISPR-Cas system cleaves bacteriophage RNA
    (Tandfonline, 2018-10-02) Millen, Anne M.; Tremblay, Denise; Moineau, Sylvain; Samson, Julie; Magadán, Alfonso H.; Rousseau, Geneviève M.; Romero, Dennis A.
    CRISPR-Cas defends microbial cells against invading nucleic acids including viral genomes. Recent studies have shown that type III-A CRISPR-Cas systems target both RNA and DNA in a transcriptiondependent manner. We previously found a type III-A system on a conjugative plasmid in Lactococcus lactis which provided resistance against virulent phages of the Siphoviridae family. Its naturally occurring spacers are oriented to generate crRNAs complementary to target phage mRNA, suggesting transcription-dependent targeting. Here, we show that only constructs whose spacers produce crRNAs complementary to the phage mRNA confer phage resistance in L. lactis. In vivo nucleic acid cleavage assays showed that cleavage of phage dsDNA genome was not detected within phage-infected L. lactis cells. On the other hand, Northern blots indicated that the lactococcal CRISPR-Cas cleaves phage mRNA in vivo. These results cannot exclude that single-stranded phage DNA is not being targeted, but phage DNA replication has been shown to be impaired.
  • PublicationRestreint
    Genomic diversity of phages infecting probiotic strains of Lactobacillus paracasei
    (American Society for Microbiology, 2015-12-22) Mercanti, Diego J.; Tremblay, Denise; Moineau, Sylvain; Capra, María Luján; Labrie, Simon; Luján Quiberoni, Andrea del; Rousseau, Geneviève M.
    Strains of the Lactobacillus casei group have been extensively studied because some are used as probiotics in foods. Conversely, their phages have received much less attention. We analyzed the complete genome sequences of five L. paracasei temperate phages: CL1, CL2, iLp84, iLp1308, and iA2. Only phage iA2 could not replicate in an indicator strain. The genome lengths ranged from 34,155 bp (iA2) to 39,474 bp (CL1). Phages iA2 and iLp1308 (34,176 bp) possess the smallest genomes reported, thus far, for phages of the L. casei group. The GC contents of the five phage genomes ranged from 44.8 to 45.6%. As observed with many other phages, their genomes were organized as follows: genes coding for DNA packaging, morphogenesis, lysis, lysogeny, and replication. Phages CL1, CL2, and iLp1308 are highly related to each other. Phage iLp84 was also related to these three phages, but the similarities were limited to gene products involved in DNA packaging and structural proteins. Genomic fragments of phages CL1, CL2, iLp1308, and iLp84 were found in several genomes of L. casei strains. Prophage iA2 is unrelated to these four phages, but almost all of its genome was found in at least four L. casei strains. Overall, these phages are distinct from previously characterized Lactobacillus phages. Our results highlight the diversity of L. casei phages and indicate frequent DNA exchanges between phages and their hosts.
  • PublicationRestreint
    Peptidoglycan hydrolase fusions maintain their parental specificities
    (American Society for Microbiology, 2006-04-01) Donovan, David M.; Moineau, Sylvain; Dong, Shengli; Rousseau, Geneviève M.; Garrett, Wes; Pritchard, David G.
    The increased incidence of bacterial antibiotic resistance has led to a renewed search for novel antimicrobials. Avoiding the use of broad-range antimicrobials through the use of specific peptidoglycan hydrolases (endolysins) might reduce the incidence of antibiotic-resistant pathogens worldwide. Staphylococcus aureus and Streptococcus agalactiaeare human pathogens and also cause mastitis in dairy cattle. The ultimate goal of this work is to create transgenic cattle that are resistant to mastitis through the expression of an antimicrobialprotein(s) in their milk. Toward this end, two novel antimicrobials were produced. The (i) full-length and (ii)182-amino-acid, C-terminally truncatedS. agalactiae bacteriophage B30 endolysins were fused to the mature lysostaphin protein of Staphylococcus simulans. Both fusions display lytic specificity for streptococcal pathogensandS. aureus. The full lytic ability of the truncated B30 protein also suggests that the SH3b domain at the Cterminus is dispensable. The fusions are active in a milk-like environment. They are also active against some lactic acid bacteria used to make cheese and yogurt, but their lytic activity is destroyed by pasteurization (63°Cfor 30 min). Immunohistochemical studies indicated that the fusion proteins can be expressed in cultured mammalian cells with no obvious deleterious effects on the cells, making it a strong candidate for use in future transgenic mice and cattle. Since the fusion peptidoglycan hydrolase also kills multiple human pathogens, it also may prove useful as a highly selective, multipathogen-targeting antimicrobial agent that could potentially reduce the use of broad-range antibiotics in fighting clinical infections.
  • PublicationRestreint
    Targeted genome editing of virulent phages using CRISPR-Cas9
    (2018-01-05) Lemay, Marie-Laurence; Moineau, Sylvain; Renaud, Ariane; Rousseau, Geneviève M.
    This protocol describes a straightforward method to generate specific mutations in the genome of strictly lytic phages. Briefly, a targeting CRISPR-Cas9 system and a repair template suited for homologous recombination are provided inside a bacterial host, here the Gram-positive model Lactococcus lactis MG1363. The CRISPR-Cas9 system is programmed to cleave a specific region present on the genome of the invading phage, but absent from the recombination template. The system either triggers the recombination event or exerts the selective pressure required to isolate recombinant phages. With this methodology, we generated multiple gene knockouts, a point mutation and an insertion in the genome of the virulent lactococcal phage p2. Considering the broad host range of the plasmids used in this protocol, the latter can be extrapolated to other phage-host pairs.
  • PublicationAccès libre
    Characterization of two polyvalent phages infecting Enterobacteriaceae
    (Nature Publishing Group, 2017-01-16) Hamdi, Sana; Tremblay, Denise; Moineau, Sylvain; Labrie, Simon; Rousseau, Geneviève M.; Kourda, Rim Saïed; Ben Slama, Karim
    Bacteriophages display remarkable genetic diversity and host specificity. In this study, we explore phages infecting bacterial strains of the Enterobacteriaceae family because of their ability to infect related but distinct hosts. We isolated and characterized two novel virulent phages, SH6 and SH7, using a strain of Shigella flexneri as host bacterium. Morphological and genomic analyses revealed that phage SH6 belongs to the T1virus genus of the Siphoviridae family. Conversely, phage SH7 was classified in the T4virus genus of the Myoviridae family. Phage SH6 had a short latent period of 16 min and a burst size of 103 ± 16 PFU/infected cell while the phage SH7 latent period was 23 min with a much lower burst size of 26 ± 5 PFU/infected cell. Moreover, phage SH6 was sensitive to acidic conditions (pH < 5) while phage SH7 was stable from pH 3 to 11 for 1 hour. Of the 35 bacterial strains tested, SH6 infected its S. flexneri host strain and 8 strains of E. coli. Phage SH7 lysed additionally strains of E. coli O157:H7, Salmonella Paratyphi, and Shigella dysenteriae. The broader host ranges of these two phages as well as their microbiological properties suggest that they may be useful for controlling bacterial populations.
  • PublicationAccès libre
    Phage resistance in lactic acid bacteria
    (Nova Science Publishers, Inc, 2012-01-01) Moineau, Sylvain; Deveau, Hélène; Rousseau, Geneviève M.
    Lactic Acid Bacteria (LAB) are used in a variety of industrial fermentation processes because of their ability to convert a variety of substrates into complex products. Any technological process that relies on bacterial fermentation is vulnerable to bacteriophage infections. This chapter describes the relationship between bacteriophages and their LAB hosts in the context of the food fermentation industry. Specifically, we highlight the most significant antiviral mechanisms in LAB. The primary focus will be given to LAB used by the dairy industry because it has openly acknowledged the problem of phage infections and has teamed up with academia and starter cultures companies to develop natural and engineered phage resistance systems to curtail the propagation of diverse phages.
  • PublicationAccès libre
    Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins
    (Nature Publishing Group, 2018-07-25) Moineau, Sylvain; Hynes, Alexander; Loehr, Jérémy; Goulet, Adeline; Agudelo, Daniel; Amigues, Béatrice; Doyon, Yannick; Rousseau, Geneviève M.; Romero, Dennis A.; Fremaux, Christophe; Horvath, Philippe; Cambillau, Christian
    CRISPR-Cas systems are bacterial anti-viral systems, and bacterial viruses (bacteriophages, phages) can carry anti-CRISPR (Acr) proteins to evade that immunity. Acrs can also fine-tune the activity of CRISPR-based genome-editing tools. While Acrs are prevalent in phages capable of lying dormant in a CRISPR-carrying host, their orthologs have been observed only infrequently in virulent phages. Here we identify AcrIIA6, an Acr encoded in 33% of virulent Streptococcus thermophilus phage genomes. The X-ray structure of AcrIIA6 displays some features unique to this Acr family. We compare the activity of AcrIIA6 to those of other Acrs, including AcrIIA5 (also from S. thermophilus phages), and characterize their effectiveness against a range of CRISPR-Cas systems. Finally, we demonstrate that both Acr families from S. thermophilus phages inhibit Cas9-mediated genome editing of human cells.
  • PublicationRestreint
    Streptococcus thermophilus bacteriophages
    (Elsevier Applied Science, 2010-04-14) Luján Quiberoni, Andrea del; Moineau, Sylvain; Rousseau, Geneviève M.; Ackermann, Hans-W.; Reinheimer, Jorge Alberto
    At least 345 bacteriophages infecting Streptococcus thermophilus starter cultures have been isolated; general characteristics include high thermal resistance, short latent periods and large burst size. Phages with such characteristics are primed to thrive in the cheese making environment, lysing bacterial cultures and generating low-quality fermented products. All S. thermophilus phages isolated to date are members of the Siphoviridae family and the Caudovirales order and appear to constitute a polythetic phage species comprising two large groups, cos- and pac-types, based on the mode of DNA packaging. Comparative analyses have shown that S. thermophilus phage genomes are similarly organized into distinct modular regions and allow the detection of a core genome region. Several PCR-based techniques have been designed to detect them in cheese whey and milk samples. Similar S. thermophilus phages are globally distributed and endemic in specific dairy environments. The biogeography of S. thermophilus phages reinforces their current classification.
  • PublicationAccès libre
    A mutation in the methionine aminopeptidase gene provides phage resistance in Streptococcus thermophilus
    (Nature Publishing Group, 2019-09-25) Tremblay, Denise; Mosterd, Cas; Loignon, Stéphanie; Moineau, Sylvain; Dupuis, Marie-Ève; Labrie, Simon; Rousseau, Geneviève M.; Romero, Dennis A.; Desjardins, Philippe; Horvath, Philippe; Fremaux, Christophe
    Streptococcus thermophilus is a lactic acid bacterium widely used by the dairy industry for the manufacture of yogurt and specialty cheeses. It is also a Gram-positive bacterial model to study phagehost interactions. CRISPR-Cas systems are one of the most prevalent phage resistance mechanisms in S. thermophilus. Little information is available about other host factors involved in phage replication in this food-grade streptococcal species. We used the model strain S. thermophilus SMQ-301 and its virulent phage DT1, harboring the anti-CRISPR protein AcrIIA6, to show that a host gene coding for a methionine aminopeptidase (metAP) is necessary for phage DT1 to complete its lytic cycle. A single mutation in metAP provides S. thermophilus SMQ-301 with strong resistance against phage DT1. The mutation impedes a late step of the lytic cycle since phage adsorption, DNA replication, and protein expression were not afected. When the mutated strain was complemented with the wild-type version of the gene, the phage sensitivity phenotype was restored. When this mutation was introduced into other S. thermophilus strains it provided resistance against cos-type (Sf21dt1virus genus) phages but replication of pac-type (Sf11 virus genus) phages was not afected. The mutation in the gene coding for the MetAP induces amino acid change in a catalytic domain conserved across many bacterial species. Introducing the same mutation in Streptococcus mutans also provided a phage resistance phenotype, suggesting the wide-ranging importance of the host methionine aminopeptidase in phage replication.