Skills (4)
Research experience
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Jan 2006–
Feb 2013Research: University College Cork
University College Cork · Department of MicrobiologyIreland (Republic of Ireland) · CorkIsolation and characterisation of phages of lactic acid bacteria. Defining the interactions between these phages and their hosts. Comparative genomic analysis of phages and LAB and evolutionary assessment.
Publications (17) View all
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Article: Investigation of the relationship between lactococcal host cell wall polysaccharide genotype and 936 phage receptor binding protein phylogeny.
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ABSTRACT: Comparative genomics of eleven lactococcal 936-type phages combined with host range analysis allowed sub-grouping of these phage genomes, particularly with respect to their encoded receptor-binding proteins. The so-called pellicle or cell wall polysaccharide of L. lactis, which has been implicated as a host receptor of (certain) 936-type phages, is specified by a large gene cluster, which among different lactococcal strains contains highly conserved regions as well as regions of diversity. The regions of diversity within this cluster on the genomes of lactococcal strains MG1363, SK11, IL1403, KF147, CV56 and UC509.9 were used for the development of a multiplex PCR system to identify the pellicle genotype of lactococcal strains used in this study. The resulting comparative analysis revealed an apparent correlation between the pellicle genotype of a given host strain and the host range of tested 936-type phages. Such a correlation would allow prediction of the intrinsic 936-type phage-sensitivity of a particular lactococcal strain, and substantiates the notion that the lactococcal pellicle polysaccharide represents the receptor for (certain) 936-type phages, while also partially explaining the molecular reasons behind the observed narrow host range of such phages.Applied and environmental microbiology 05/2013; · 3.69 Impact Factor -
Article: Molecular analysis of lactococcal phages Q33 and BM13: Identification of a new P335 subgroup.
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ABSTRACT: Lactococcal dairy starter strains are under constant threat from phages in dairy fermentation facilities, especially by members of the so-called 936, P335, and c2 species. Among these three phage groups, members of the P335 species represent the most genetically diverse. Here we present the complete genome sequence of two P335-type phages, Q33 and BM13, isolated in North America and representing a novel lineage within this phage group. The Q33 and BM13 genomes exhibit homology not only to P335-type, but also elements of the 936-type phage sequences. These two phage genomes also have close relatedness to phages infecting Enterococcus and Clostridium, a heretofore unknown feature among lactococcal P335 phages. The Q33 and BM13 genomes are organised in functionally related clusters with genes encoding functions such as DNA replication and packaging, morphogenesis and host cell lysis. Electron micrographic analysis of the two phages highlights the presence of a baseplate being more reminiscent of the baseplate of 936 phages than that the majority of members of the P335 group, with the exception of r1t and LC3.Applied and environmental microbiology 05/2013; · 3.69 Impact Factor -
Article: Viral infection modulation and neutralization by camelid nanobodies
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ABSTRACT: Lactococcal phages belong to a large family of Siphoviridae and infect Lactococcus lactis, a Gram-positive bacterium used in com-mercial dairy fermentations. These phages are believed to recog-nize and bind specifically to pellicle polysaccharides covering the entire bacterium. The phage TP901-1 baseplate, located at the tip of the tail, harbors 18 trimeric receptor binding proteins (RBPs) promoting adhesion to a specific lactococcal strain. Phage TP901-1 adhesion does not require major conformational changes or Ca 2+ , which contrasts other lactococcal phages. Here, we produced and characterized llama nanobodies raised against the purified base-plate and the Tal protein of phage TP901-1 as tools to dissect the molecular determinants of phage TP901-1 infection. Using a set of complementary techniques, surface plasmon resonance, EM, and X-ray crystallography in a hybrid approach, we identified binders to the three components of the baseplate, analyzed their affinity for their targets, and determined their epitopes as well as their functional impact on TP901-1 phage infectivity. We determined the X-ray structures of three nanobodies in complex with the RBP. Two of them bind to the saccharide binding site of the RBP and are able to fully neutralize TP901-1 phage infectivity, even after 15 passages,. These results provide clear evidence for a practical use of nanobodies in circumventing lactococcal phages viral infection in dairy fermentation. T ailed bacteriophages (Caudovirales order) typically possess a tail distal machinery used to recognize the host with high specificity as well as ensure genome delivery. The Caudovirales phylum encompasses three families: phages belonging to the Myoviridae family possess a contractile tail (e.g., T4) (1), and Podoviridae phages have a short tail (e.g., P22) (2, 3), whereas members of the Siphoviridae bear a long noncontractile tail such as HK97 (4), SPP1 (5), or TP901-1 (6–8). The siphophages distal machinery can be described as a straight tail tip, a morphology identified in phages binding to a membrane protein as receptor, such as Escherichia coli phage-λ (9, 10), Bacillus subtilis phage SPP1 (5, 11, 12), or Lactococcus lactis phage C2 (13). Phages recognizing and binding to saccha-ridic receptors possess a massive organelle, the baseplate, car-rying a large number (12 or more) of antireceptor proteins, also called receptor binding proteins (RBPs) (6, 14, 15). This setting probably provides avidity of multiple receptor binding events to compensate for the moderate affinity of a single saccharidic re-ceptor for an individual RBP. In recent years, we have reported on the structures of the RBPs and the baseplate of two phages, p2 and TP901-1, which infect the Gram + bacterium L. lactis, and we have identified their different strategies used to ensure infection (6, 8, 14–19). Although Ca 2+ ions trigger large conformational changes of phage p2 baseplate to orientate the RBPs to the host (15), the RBPs of phage TP901-1 baseplate point to the appropriate direction without the need for conformational change or Ca 2+ requirement (6). These structural data were confirmed and extended to members of the 936 (p2) and P335 (TP901-1) lactococcal phages groups using in vivo infection experiments (6). We hypothesized that, as in the case of phage SPP1 (5, 20), host adhesion would generate a signal, which after propagation along the tail, would promote opening of the portal complex and ejection of the dsDNA genome out of the capsid. The origin of such a signal should reside in the baseplate, be-cause it is in direct contact with the host cell envelope. Although the large conformational change of p2 baseplate is an obvious candidate for initiating such a signal, the case of TP901-1 is less straightforward. We have proposed that subtle conformational changes occur on binding of a TP901-1 tripod [a complex of three RBP trimers linked to an upper baseplate protein (BppU) holder] (6) to the host and thus, may generate the initial signal. Here, we designed an approach, based on the use of camelid antibodies fragments, nanobodies, or variable domain of heavy-chain anti-body (vHH) (21, 22), to explore this hypothesis. These nano-bodies have been found to be able to neutralize phages and viruses (23–25) by blocking their RBDs (14, 15, 26). The TP901-1 baseplate is composed of four proteins: the two proteins aligned along the phage axis, Dit (distal tail) and Tal (tail-associated lysozyme), and the two peripheral proteins, BppU and lower Bpp (BppL) (the RBP) (8). The TP901-1 complete base-plate comprises 6 Dit, 3 Tal, 18 BppU, and 54 BppL. We have expressed a baseplate of TP901-1 (BP TP901-1) containing all of the baseplate components except Tal (i.e., a complex of 78 proteins and 1.8 MDa) (6, 17) (Fig. 1). We have also expressed and purified subcomponents of the baseplate: Dit alone as a monomer, the trimer of BppL (the RBP) (Fig. 1) (16, 27), a complex of three BppU and nine BppL/RBP (the long tripod) (6, 17), and theProceedings of the National Academy of Sciences 03/2013; · 9.68 Impact Factor -
Article: The lactococcal phages Tuc2009 and TP901-1 incorporate two alternate forms of their tail fibre into their virions for infection specialization.
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ABSTRACT: Lactococcal phages Tuc2009 and TP901-1 possess a conserved tail fibre, called a Tail-Associated Lysin (referred to as Tal2009 for Tuc2009, and Tal901-1 for TP901-1), suspended from their tail tips that projects a Peptidoglycan-Hydrolase (PGH) domain towards a potential host bacterium. Tal2009 and Tal901-1 can undergo proteolytic processing mid-protein at the glycine-rich sequence GG(S/N)SGGG, removing their C-terminal structural lysin. In this study, we show that the PGH of these Tal proteins is an M23 peptidase which exhibits D-Ala-D-Asp endopeptidase activity, and that this activity is required for efficient infection of stationary phase cells. Interestingly, the observed proteolytic processing of Tal2009 and Tal901-1 facilitates increased host adsorption efficiencies of the resulting phages. This represents, to the best of our knowledge, the first example of tail fibre proteolytic processing that results in a heterogeneous population of two phage types. Phages which possess a full-length tail fibre, or a truncated derivative, are better adapted to efficiently infect cells with extensively cross-linked cell wall, or infect with increased host-adsorption efficiencies, respectively.Journal of Biological Chemistry 01/2013; · 4.77 Impact Factor -
Dataset: Complete Genome of Lactococcus lactis subsp. cremoris UC509.9, Host for a Model Lactococcal P335 Bacteriophage
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ABSTRACT: Here, we report the complete genome of Lactococcus lactis subsp. cremoris UC509.9, an Irish dairy starter. The circular chromo-some of L. lactis UC509.9 represents the smallest among those of the sequenced lactococcal strains, while its large complement of eight plasmids appears to be a reflection of its adaptation to the dairy environment. Citation Ainsworth S, Zomer A, de Jager V, Bottacini F, van Hijum SAFT, Mahony J, van Sinderen D. 2013. Complete genome of Lactococcus lactis subsp. cremoris UC509.9, host for a model lactococcal P335 bacteriophage. Genome Announc. 1(1):e00119-12. doi:10.1128/genomeA.00119-12. Copyright © 2013 Ainsworth et al. This is an open-access article distributed under the terms of the Attribution 3.0 Unported Creative Commons License. Address correspondence to Douwe van Sinderen, d.vansinderen@ucc.ie. L actococcus lactis strains are used extensively worldwide for the production of fermented dairy products. Bacteriophage (phage) attack during this fermentation process can lead to slow or failed fermentations and is therefore of major economic con-cern (1). L. lactis subsp. cremoris UC509 is an Irish cheddar starter strain and is the lysogenic host of the model P335-type phage Tuc2009 (2–6). L. lactis UC509.9, whose genome sequence is pre-sented here, is a prophage-cured Tuc2009-sensitive derivative of UC509 (7). While lactococcal phages are subject to intensive scientific scrutiny, the specific interactions with their hosts are poorly un-derstood. To further our understanding regarding the molecular interplay between Tuc2009 and its host, we sequenced the genome of L. lactis UC509.9. Sequencing was performed by Agencourt Bioscience (Beverly, MA) and Macrogen (Seoul, Republic of Ko-rea) using a combination of 454 sequencing of a 3-kb fragment library using Roche standard procedures and of Sanger sequenc-ing of a 36-kb insert library followed by homopolymer tract cor-rection using Illumina sequencing. Initial sequence assembly was performed using GSassembler (Roche). Gap closure and quality improvements were performed by Sanger sequencing of gap-closing PCR products as suggested by Projector 2 (8) with the Staden package (9). Homopolymer tract single nucleotide poly-morphisms (SNPs) were detected and corrected using Robust Variant detection (ROVAR) (V. de Jager, B. Renckens, R. J. Siezen, and S. A. F. T. van Hijum, unpublished data [https://trac.nbic.nl /rovar/]) applied to Illumina sequencing data as described previ-ously (10), resulting in a 200-fold coverage of the genome. Pu-tative protein-encoding genes were identified using Prodigal version 2.0 (11). The results were inspected using Artemis (12), with manual checking and editing using BLASTP, Pfam (13), Kyoto Encyclopedia of Genes and Genomes (KEGG) (14), and Clusters of Orthologous Groups (COG) databases (15). The complete genome of L. lactis UC509.9 consists of a single circular chromosome of 2,250,427 bp (35.88% GC content) plus eight plasmids: pCIS1 (4,263 bp), pCIS2 (5,961 bp), pCIS3 (6,159 bp), pCIS4 (7,045 bp), pCIS5 (11,676 bp), pCIS6 (40,285 bp), pCIS7 (53,051 bp), and pCIS8 (80,592 bp). The L. lac-tis UC509.9 genome is predicted to contain 2,066 protein-encoding genes, of which 168 are pseudogenes. Forty-three of these 168 pseudogenes are identical to those found in L. lactis subsp. cremoris SK11 (GenBank accession no. CP000425.1). The genome of L. lactis UC509.9 contains 104 transposase-encoding genes involving a total of 106,746 bp, including 42 copies of IS182 and 29 copies of IS981. The combination of the smallest lactococ-cal chromosome identified so far and the high number of trans-posons and pseudogenes suggests that the genome has undergone significant genome decay while adapting to the nutrient-rich dairy environment. A region of approximately 11 kb in size not present in other L. lactis genomes appears to be an integrated plasmid that includes the restriction-modification system ScrFII (16). The L. lactis UC509.9 plasmid complement encodes various traits for adaptation to the dairy environment, such as lactose and casein metabolism. Nucleotide sequence accession numbers. The complete chro-mosome and plasmid complement of L. lactis subsp. cremoris UC509.9 were deposited in GenBank under accession no. CP003157 (chromosome), CP003165 (pCIS1), CP003164 (pCIS2), CP003163 (pCIS3), CP003162 (pCIS4), CP003161 (pCIS5), CP003160 (pCIS6), CP003159 (pCIS7), and CP003158 (pCIS8).
