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Tremblay, Denise

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Tremblay

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Denise

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Université Laval. Faculté des sciences et de génie. Département de biochimie

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Voici les éléments 1 - 10 sur 52
  • PublicationAccès libre
    Characterization and diversity of phages infecting Aeromonas salmonicida subsp. salmonicida
    (Nature Publishing Group, 2017-08-01) Tremblay, Denise; Paquet, Valérie; Bernatchez, Alex; Moineau, Sylvain; Vincent, Antony; Charette, Steve
    Phages infecting Aeromonas salmonicida subsp. salmonicida, the causative agent of the fish disease furunculosis, have been isolated for decades but very few of them have been characterized. Here, the host range of 12 virulent phages, including three isolated in the present study, was evaluated against a panel of 65 A. salmonicida isolates, including representatives of the psychrophilic subspecies salmonicida, smithia, masoucida, and the mesophilic subspecies pectinolytica. This bacterial set also included three isolates from India suspected of being members of a new subspecies. Our results allowed to elucidate a lytic dichotomy based on the lifestyle of A. salmonicida (mesophilic or psychrophilic) and more generally, on phage types (lysotypes) for the subspecies salmonicida. The genomic analyses of the 12 phages from this study with those available in GenBank led us to propose an A. salmonicida phage pan-virome. Our comparative genomic analyses also suggest that some phage genes were under positive selection and A. salmonicida phage genomes having a discrepancy in GC% compared to the host genome encode tRNA genes to likely overpass the bias in codon usage. Finally, we propose a new classification scheme for A. salmonicida phages.
  • PublicationRestreint
    Crystal structure of the receptor-binding protein head domain from lactococcus lactis phage bIL170
    (American Society for Microbiology, 2006-09) Ricagno, Stefano; Tremblay, Denise; Campanacci, Valérie; Moineau, Sylvain; Blangy, Stéphanie; Spinelli, Silvia; Tegoni, Mariella; Cambillau, Christian
    Lactococcus lactis, a gram-positive bacterium widely used by the dairy industry, is subject to lytic phage infections. In the first step of infection, phages recognize the host saccharidic receptor using their receptor binding protein (RBP). Here, we report the 2.30-Å-resolution crystal structure of the RBP head domain from phage bIL170. The structure of the head monomer is remarkably close to those of other lactococcal phages, p2and TP901-1, despite any sequence identity with them. The knowledge of the three-dimensional structures of three RBPs gives a better insight into the module exchanges which have occurred among phages.
  • PublicationAccès libre
    Crystal structure and function of a DARPin neutralizing inhibitor of lactococcal phage TP901-1 : comparison of DARPin and camelid VHH binding mode
    (American Society for Biochemistry and Molecular Biology, Inc., 2009-09-09) Veesler, David; Tremblay, Denise; Dreier, Birgit; Moineau, Sylvain; Blangy, Stéphanie; Lichière, Julie; Spinelli, Silvia; Tegoni, Mariella; Plückthun, Andreas; Campanacci, Valérie; Cambillau, Christian
    Combinatorial libraries of designed ankyrin repeat proteins (DARPins) have been proven to be a valuable source of specific binding proteins, as they can be expressed at very high levels and are very stable. We report here the selection of DARPins directed against a macromolecular multiprotein complex, the baseplate BppU·BppL complex of the lactococcal phage TP901-1. Using ribosome display, we selected several DARPins that bound specifically to the tip of the receptor-binding protein (RBP, the BppL trimer). The three selected DARPins display high specificity and affinity in the low nanomolar range and bind with a stoichiometry of one DARPin per BppL trimer. The crystal structure of a DARPin complexed with the RBP was solved at 2.1 Å resolution. The DARPin·RBP interface is of the concave (DARPin)-convex (RBP) type, typical of other DARPin protein complexes and different from what is observed with a camelid VHH domain, which penetrates the phage p2 RBP inter-monomer interface. Finally, phage infection assays demonstrated that TP901-1 infection of Lactococcus lactis cells was inhibited by each of the three selected DARPins. This study provides proof of concept for the possible use of DARPins to circumvent viral infection. It also provides support for the use of DARPins in co-crystallization, due to their rigidity and their ability to provide multiple crystal contacts.
  • 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.
  • PublicationAccès libre
    Prophages of the genus Bifidobacterium as modulating agents of the infant gut microbiota
    (2016-07-08) Lugli, Gabriele Andrea; Tremblay, Denise; Milani, Christian; Moineau, Sylvain; Turroni, Francesca; Priego, Sabrina.; Mancabelli, Leonardo; Ward, Doyle V.; Ossiprandi, Maria Cristina; Sinderen, Douwe van; Ventura, Marco
    Phage predation is one of the key forces that shape genetic diversity in bacterial genomes. Phages are also believed to act as modulators of the microbiota composition and, consequently, as agents that drive bacterial speciation in complex bacterial communities. Very little is known about the occurrence and genetic variability of (pro)phages within the genus, a dominant bacterial group of the human infant microbiota. Here, we performed cataloguing of the predicted prophage sequences from the genomes of all currently recognized bifidobacterial type strains. We analysed their genetic diversity and deduced their evolutionary development, thereby highlighting an intriguing origin. Furthermore, we assessed infant gut microbiomes for the presence of (pro)phage sequences and found compelling evidence that these viral elements influence the composition of bifidobacterial communities in the infant gut microbiota
  • PublicationAccès libre
    7-Deazaguanine modifications protect phage DNA from host restriction systems
    (Nature Publishing Group, 2019-11-29) Hutinet, Geoffrey; Tremblay, Denise; Kot, Witold; Moineau, Sylvain; Cui, Liang; Hillebrand, Roman; Balamkundu, Seetharamsingh; Gnanakalai, Shanmugavel; Neelakandan, Ramesh; Carstens, Alexander B.; Liu, Chuan Fa; Jacobs-Sera, Deborah; Sassanfar, Mandana; Lee, Yan-Jiun; Weigele, Peter; Hatfull, Graham F.; Dedon, Peter C.; Hansen, Lars H.; De Crécy-Lagard, Valérie
    Genome modifications are central components of the continuous arms race between viruses and their hosts. The archaeosine base (G+), which was thought to be found only in archaeal tRNAs, was recently detected in genomic DNA of Enterobacteria phage 9g and was proposed to protect phage DNA from a wide variety of restriction enzymes. In this study, we identify three additional 2′-deoxy-7-deazaguanine modifications, which are all intermediates of the same pathway, in viruses: 2′-deoxy-7-amido-7-deazaguanine (dADG), 2′-deoxy-7-cyano-7-deazaguanine (dPreQ0) and 2′-deoxy-7- aminomethyl-7-deazaguanine (dPreQ1). We identify 180 phages or archaeal viruses that encode at least one of the enzymes of this pathway with an overrepresentation (60%) of viruses potentially infecting pathogenic microbial hosts. Genetic studies with the Escherichia phage CAjan show that DpdA is essential to insert the 7-deazaguanine base in phage genomic DNA and that 2′-deoxy-7-deazaguanine modifications protect phage DNA from host restriction enzymes.
  • PublicationRestreint
    Receptor-binding protein of lactococcus lactis phages : identification and characterization of the saccharide receptor-binding site
    (American Society for Microbiology, 2006-03-17) Tremblay, Denise; Tegoni, Mariella; Labrie, Steve; Spinelli, Silvia; Moineau, Sylvain; Campanacci, Valérie; Blangy, Stéphanie; Huyghe, Céline; Desmyter, Aline; Cambillau, Christian
    Phage p2, a member of the lactococcal 936 phage species, infects Lactococcus lactis strains by binding initially to specific carbohydrate receptors using its receptor-binding protein (RBP). The structures of p2 RBP, a homotrimeric protein composed of three domains, and of its complex with a neutralizing llama VH domain (VHH5) have been determined (S. Spinelli, A. Desmyter, C. T. Verrips, H. J. de Haard, S. Moineau, and C. Cambillau, Nat. Struct. Mol. Biol. 13:85–89, 2006). Here, we show that VHH5 was able to neutralize 12 of 50 lactococcal phages belonging to the 936 species. Moreover, escape phage mutants no longer neutralized by VHH5 were isolated from 11 of these phages. All of the mutations (but one) cluster in the RBP/VHH5 interaction surface that delineates the receptor-binding area. A glycerol molecule, observed in the 1.7-Å resolution structure of RBP, was found to bind tightly (Kd = 0.26 M) in a crevice located in this area. Other saccharides bind RBP with comparable high affinity. These data prove the saccharidic nature of the bacterial receptor recognized by phage p2 and identify the position of its binding site in the RBP head domain.
  • PublicationAccès libre
    Structure of lactococcal phage p2 baseplate and its mechanism of activation
    (National Academy of Sciences, 2010-04-13) Sciara, Giuliano; Tremblay, Denise; Bebeacua, Cecilia; Moineau, Sylvain; Bron, Patrick; Ortiz-Lombardia, Miguel; Lichière, Julie; Heel, Marin van; Campanaccia, Valérie; Cambillau, Christian
    Siphoviridae is the most abundant viral family on earth which infects bacteria as well as archaea. All known siphophages infecting gram+ Lactococcus lactis possess a baseplate at the tip of their tail involved in host recognition and attachment. Here, we report analysis of the p2 phage baseplate structure by X-ray crystallography and electron microscopy and propose a mechanism for the baseplate activation during attachment to the host cell. This ∼1 MDa, Escherichia coli-expressed baseplate is composed of three protein species, including six trimers of the receptor-binding protein (RBP). RBPs host-recognition domains point upwards, towards the capsid, in agreement with the electron-microscopy map of the free virion. In the presence of Ca2+, a cation mandatory for infection, the RBPs rotated 200° downwards, presenting their binding sites to the host, and a channel opens at the bottom of the baseplate for DNA passage. These conformational changes reveal a novel siphophage activation and host-recognition mechanism leading ultimately to DNA ejection.
  • PublicationRestreint
    Structure, adsorption to host, and infection mechanism of virulent lactococcal phage p2
    (American Society for Microbiology, 2013-09-11) Bebeacua, Cecilia; Tremblay, Denise; Moineau, Sylvain; Farenc, Carine; Chapot-Chartier, Marie-Pierre; Sadovskaya, Irina; Heel, Marin van; Veesler, David; Cambillau, Christian
    Lactococcal siphophages from the 936 and P335 groups infect the Gram-positive bacterium Lactococcus lactis using receptor binding proteins (RBPs) attached to their baseplate, a large multiprotein complex at the distal part of the tail. We have previously reported the crystal and electron microscopy (EM) structures of the baseplates of phages p2 (936 group) and TP901-1 (P335 group) as well as the full EM structure of the TP901-1 virion. Here, we report the complete EM structure of siphophage p2, including its capsid, connector complex, tail, and baseplate. Furthermore, we show that the p2 tail is characterized by the presence of protruding decorations, which are related to adhesins and are likely contributed by the major tail protein C-terminal domains. This feature is reminiscent of the tail of Escherichia coli phage λ and Bacillus subtilis phage SPP1 and might point to a common mechanism for establishing initial interactions with their bacterial hosts. Comparative analyses showed that the architecture of the phage p2 baseplate differs largely from that of lactococcal phage TP901-1. We quantified the interaction of its RBP with the saccharidic receptor and determined that specificity is due to lower koff values of the RBP/saccharidic dissociation. Taken together, these results suggest that the infection of L. lactis strains by phage p2 is a multistep process that involves reversible attachment, followed by baseplate activation, specific attachment of the RBPs to the saccharidic receptor, and DNA ejection.
  • 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.