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  • Journal article
    Dembek M, Stabler RA, Witney AA, Wren BW, Fairweather NFet al., 2013,

    Transcriptional analysis of temporal gene expression in germinating clostridium difficile 630 endospores

    , PLOS One, Vol: 8, ISSN: 1932-6203

    Clostridium difficile is the leading cause of hospital acquired diarrhoea in industrialised countries. Under conditions that are not favourable for growth, the pathogen produces metabolically dormant endospores via asymmetric cell division. These are extremely resistant to both chemical and physical stress and provide the mechanism by which C. difficile can evade the potentially fatal consequences of exposure to heat, oxygen, alcohol, and certain disinfectants. Spores are the primary infective agent and must germinate to allow for vegetative cell growth and toxin production. While spore germination in Bacillus is well understood, little is known about C. difficile germination and outgrowth. Here we use genome-wide transcriptional analysis to elucidate the temporal gene expression patterns in C. difficile 630 endospore germination. We have optimized methods for large scale production and purification of spores. The germination characteristics of purified spores have been characterized and RNA extraction protocols have been optimized. Gene expression was highly dynamic during germination and outgrowth, and was found to involve a large number of genes. Using this genome-wide, microarray approach we have identified 511 genes that are significantly up- or down-regulated during C. difficile germination (p≤0.01). A number of functional groups of genes appeared to be co-regulated. These included transport, protein synthesis and secretion, motility and chemotaxis as well as cell wall biogenesis. These data give insight into how C. difficile re-establishes its metabolism, re-builds the basic structures of the vegetative cell and resumes growth.

  • Journal article
    Leen EN, Kwok KYR, Birtley JR, Simpson PJ, Subba-Reddy CV, Chaudhry Y, Sosnovtsev SV, Green KY, Prater SN, Tong M, Young JC, Chung LMW, Marchant J, Roberts LO, Kao CC, Matthews S, Goodfellow IG, Curry Set al., 2013,

    Structures of the Compact Helical Core Domains of Feline Calicivirus and Murine Norovirus VPg Proteins

    , JOURNAL OF VIROLOGY, Vol: 87, Pages: 5318-5330, ISSN: 0022-538X
  • Journal article
    Aurass P, Schlegel M, Metwally O, Harding CR, Schroeder GN, Frankel G, Flieger Aet al., 2013,

    The Legionella pneumophila Dot/Icm-secreted Effector PlcC/CegC1 Together with PlcA and PlcB Promotes Virulence and Belongs to a Novel Zinc Metallophospholipase C Family Present in Bacteria and Fungi

    , JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 288, Pages: 11080-11092
  • Journal article
    Figueira R, Watson KG, Holden DW, Helaine Set al., 2013,

    Identification of salmonella pathogenicity island-2 type III secretion system effectors involved in intramacrophage replication of S. enterica serovar typhimurium: implications for rational vaccine design

    , mBio, Vol: 4, ISSN: 2161-2129

    Salmonella enterica serovars cause severe diseases in humans, such as gastroenteritis and typhoid fever. The development of systemic disease is dependent on a type III secretion system (T3SS) encoded by Salmonella pathogenicity island-2 (SPI-2). Translocation of effector proteins across the Salmonella-containing vacuole, via the SPI-2 T3SS, enables bacterial replication within host cells, including macrophages. Here, we investigated the contribution of these effectors to intramacrophage replication of Salmonella enterica serovar Typhimurium using Fluorescence Dilution, a dual-fluorescence tool which allows direct measurement of bacterial replication. Of 32 strains, each carrying single mutations in genes encoding effectors, 10 (lacking sifA, sseJ, sopD2, sseG, sseF, srfH, sseL, spvD, cigR, or steD) were attenuated in replication in mouse bone marrow-derived macrophages. The replication profiles of strains combining deletions in effector genes were also investigated: a strain lacking the genes sseG, sopD2, and srfH showed an increased replication defect compared to single-mutation strains and was very similar to SPI-2 T3SS-deficient bacteria with respect to its replication defect. This strain was substantially attenuated in virulence in vivo and yet retained intracellular vacuole integrity and a functional SPI-2 T3SS. Moreover, this strain was capable of SPI-2 T3SS-mediated delivery of a model antigen for major histocompatibility complex (MHC) class I-dependent T-cell activation. This work establishes a basis for the use of a poly-effector mutant strain as an attenuated vaccine carrier for delivery of heterologous antigens directly into the cytoplasm of host cells.IMPORTANCE Live attenuated strains of Salmonella enterica serotype Typhi have generated much interest in the search for improved vaccines against typhoid fever and as vaccine vectors for the delivery of heterologous antigens. A promising vaccine candidate is the ΔaroC ΔssaV S. Typhi strain, whic

  • Journal article
    Permpoonpattana P, Phetcharaburanin J, Mikelsone A, Dembek M, Tan S, Brisson M-C, La Ragione R, Brisson AR, Fairweather N, Hong HA, Cutting SMet al., 2013,

    Functional Characterization of Clostridium difficile Spore Coat Proteins

    , JOURNAL OF BACTERIOLOGY, Vol: 195, Pages: 1492-1503, ISSN: 0021-9193
  • Journal article
    Sheppard C, James E, Barton G, Matthews S, Severinov K, Wigneshweraraj Set al., 2013,

    A non-bacterial transcription factor inhibits bacterial transcription by a multipronged mechanism

    , RNA BIOLOGY, Vol: 10, Pages: 495-501, ISSN: 1547-6286
  • Journal article
    Helaine S, Holden DW, 2013,

    Heterogeneity of intracellular replication of bacterial pathogens

    , CURRENT OPINION IN MICROBIOLOGY, Vol: 16, Pages: 184-191, ISSN: 1369-5274
  • Journal article
    Hachani A, Lossi NS, Filloux A, 2013,

    A visual assay to monitor T6SS-mediated bacterial competition

    , Jove-Journal of Visualized Experiments, ISSN: 1940-087X

    Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.The data available emphasized a numerical approach to the bacterial competition assay

  • Journal article
    Lossi NS, Manoli E, Foerster A, Dajani R, Pape T, Freemont P, Filloux Aet al., 2013,

    The HsiB1C1 (TssB-TssC) Complex of the Pseudomonas aeruginosa Type VI Secretion System Forms a Bacteriophage Tail Sheathlike Structure

    , JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 288, Pages: 7536-7548, ISSN: 0021-9258
  • Journal article
    Firdessa R, Berg S, Hailu E, Schelling E, Gumi B, Erenso G, Gadisa E, Kiros T, Habtamu M, Hussein J, Zinsstag J, Robertson BD, Ameni G, Lohan AJ, Loftus B, Comas I, Gagneux S, Tschopp R, Yamuah L, Hewinson G, Gordon SV, Young DB, Aseffa Aet al., 2013,

    Mycobacterial Lineages Causing Pulmonary and Extrapulmonary Tuberculosis, Ethiopia

    , Emerging Infectious Diseases, Vol: 19, Pages: 460-463, ISSN: 1080-6059

    Molecular typing of 964 specimens from patients in Ethiopia with lymph node or pulmonary tuberculosis showed a similar distribution of Mycobacterium tuberculosis strains between the 2 disease manifestations and a minimal role for M. bovis. We report a novel phylogenetic lineage of M. tuberculosis strongly associated with the Horn of Africa.

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