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  • Conference paper
    Bernal P, Civantos C, Filloux A, Llamas MAet al., 2015,

    Type VI secretion in the plant growth promoting rhizobacteria Pseudomonas putida

    , FEMS Microbiology Congress 2015

    BackgroundBacterial type VI secretion systems (T6SSs) are recently discovered nanomachines used to inject effectors into prokaryotic or eukaryotic cells. Therefore, T6SSs are involved in both inter-bacterial competition and bacterial pathogenesis.ObjectivesThe aim is the study of the T6SS of Pseudomonas putida a soil bacterium with the capacity to colonise the root of crop plants. The colonisation by this bacterium provides growth advantages to the plant and, importantly, protection against plant pathogens. This makes P. putida a relevant biocontrol agent. Since T6SS is mainly used by environmental bacteria for interbacterial competition, one might speculate that T6SSs play a relevant role in the biocontrol properties of P. putida. Methods• in silico analysis of P. putida KT2440 genome • Competition assays to determine H1-T6SS activity and for the identification of H1-T6SS targets.• Regulatory studies: qRT-PCR, transcriptional fusionsConclusionsThe in silico analysis has revealed the existence of three putative T6SSs (H1, H2, and H3). The clusters contain the genes encoding the conserved core components and some accessories, including regulatory proteins and toxins-immunity pairs. Additional T6SS-related genes are found scattered on the chromosome.By competition assays we have determined that H1-T6SS is active and that mutants in H1-T6SS structural components lack the ability to kill model prey strains. Moreover, the system can be used to kill serious phytopathogens such as Pseudomonas syringae in in vitro assays. Interestingly, the H1-T6SS is induced in stationary phase and controlled by the global regulators RetS and GacS-GacA, and by two alternative sigma factors, RpoS and RpoN.
  • Journal article
    Sana TG, Baumann C, Merdes A, Soscia C, Rattei T, Hachani A, Jones C, Bennett KL, Filloux A, Superti-Furga G, Voulhoux R, Bleves Set al., 2015,

    Internalization of Pseudomonas aeruginosa Strain PAO1 into Epithelial Cells Is Promoted by Interaction of a T6SS Effector with the Microtubule Network.

    , mBio, Vol: 6, ISSN: 2150-7511

    Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first example of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction. IMPORTANCE: Innate immunity and specifically professional phagocytic cells are key determinants in the ability of the host to control P. aeruginosa infection. However, among various virulence strategies, includi
  • Journal article
    Garnett JA, Diallo M, Matthews SJ, 2015,

    Purification, crystallization and preliminary X-ray diffraction analysis of the Escherichia coli common pilus chaperone EcpB

    , Acta Crystallographica Section F: Structural Biology Communications, Vol: 71, Pages: 676-679, ISSN: 2053-230X

    Pili are key cell-surface components that allow the attachment of bacteria to both biological and abiotic solid surfaces, whilst also mediating interactions between themselves. In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone-usher (CU) pathway that plays a major role in both early biofilm formation and host-cell adhesion. The chaperone EcpB is involved in the biogenesis of the filament, which is composed of EcpA and EcpD. Initial attempts at crystallizing EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.4 Å resolution. These crystals belonged to the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 62.65, c = 121.14 Å and one monomer in the asymmetric unit. Molecular replacement was unsuccessful, but selenomethionine-substituted protein and heavy-atom derivatives are being prepared for phasing. The three-dimensional structure of EcpB will provide invaluable information on the subtle mechanistic differences in biogenesis between the alternative and classical CU pathways. Furthermore, this is the first time that this refolding strategy has been used to purify CU chaperones, and it could be implemented in similar systems where it has not been possible to obtain highly ordered crystals.
  • Journal article
    Messens W, Bolton D, Frankel G, Liebana E, McLauchlin J, Morabito S, Oswald E, Threlfall EJet al., 2015,

    Defining pathogenic verocytotoxin-producing <i>Escherichia coli</i> (VTEC) from cases of human infection in the European Union, 2007-2010

    , EPIDEMIOLOGY AND INFECTION, Vol: 143, Pages: 1652-1661, ISSN: 0950-2688
  • Journal article
    Couchman EC, Browne HP, Dunn M, Lawley TD, Songer JG, Hall V, Petrovska L, Vidor C, Awad M, Lyras D, Fairweather NFet al., 2015,

    Clostridium sordellii genome analysis reveals plasmid localized toxin genes encoded within pathogenicity loci

    , BMC Genomics, Vol: 16, ISSN: 1471-2164

    BackgroundClostridium sordellii can cause severe infections in animals and humans, the latter associated with trauma, toxic shock and often-fatal gynaecological infections. Strains can produce two large clostridial cytotoxins (LCCs), TcsL and TcsH, related to those produced by Clostridium difficile, Clostridium novyi and Clostridium perfringens, but the genetic basis of toxin production remains uncharacterised.ResultsPhylogenetic analysis of the genome sequences of 44 strains isolated from human and animal infections in the UK, US and Australia placed the species into four clades. Although all strains originated from animal or clinical disease, only 5 strains contained LCC genes: 4 strains contain tcsL alone and one strain contains tcsL and tcsH. Four toxin-positive strains were found within one clade. Where present, tcsL and tcsH were localised in a pathogenicity locus, similar to but distinct from that present in C. difficile. In contrast to C. difficile, where the LCCs are chromosomally localised, the C. sordellii tcsL and tcsH genes are localised on plasmids. Our data suggest gain and loss of entire toxigenic plasmids in addition to horizontal transfer of the pathogenicity locus. A high quality, annotated sequence of ATCC9714 reveals many putative virulence factors including neuraminidase, phospholipase C and the cholesterol-dependent cytolysin sordellilysin that are highly conserved between all strains studied.ConclusionsGenome analysis of C. sordellii reveals that the LCCs, the major virulence factors, are localised on plasmids. Many strains do not contain the LCC genes; it is probable that in several of these cases the plasmid has been lost upon laboratory subculture. Our data are consistent with LCCs being the primary virulence factors in the majority of infections, but LCC-negative strains may precipitate certain categories of infection. A high quality genome sequence reveals putative virulence factors whose role in virulence can be investigated.Keywords: C
  • Journal article
    Uhia I, Williams KJ, Shahrezaei V, Robertson BDet al., 2015,

    Mycobacterial growth

    , Cold Spring Harbor Perspectives in Medicine, Vol: 5, ISSN: 2157-1422

    In this work, we review progress made in understanding the molecular underpinnings of growth and division in mycobacteria, concentrating on work published since the last comprehensive review ( Hett and Rubin 2008). We have focused on exciting work making use of new time-lapse imaging technologies coupled with reporter-gene fusions and antimicrobial treatment to generate insights into how mycobacteria grow and divide in a heterogeneous manner. We try to reconcile the different observations reported, providing a model of how they might fit together. We also review the topic of mycobacterial spores, which has generated considerable discussion during the last few years. Resuscitation promoting factors, and regulation of growth and division, have also been actively researched, and we summarize progress in these areas.
  • Journal article
    Benjamin S, Williams F, Kerry L, Matthews Set al., 2015,

    NMR assignment of the immune mapped protein 1 (IMP1) homologue from Plasmodium falciparum

    , Biomolecular NMR Assignments, Vol: 9, Pages: 393-395, ISSN: 1874-2718
  • Journal article
    Hunter PJ, Shaw RK, Berger CN, Frankel G, Pink D, Hand Pet al., 2015,

    Older leaves of lettuce (Lactuca spp.) support higher levels of Salmonella enterica ser. Senftenberg attachment and show greater variation between plant accessions than do younger leaves

    , FEMS Microbiology Letters, Vol: 362, ISSN: 0378-1097

    Salmonella can bind to the leaves of salad crops including lettuce and survive for commercially relevant periods. Previous studies have shown that younger leaves are more susceptible to colonization than older leaves and that colonization levels are dependent on both the bacterial serovar and the lettuce cultivar. In this study, we investigated the ability of two Lactuca sativa cultivars (Saladin and Iceberg) and an accession of wild lettuce (L. serriola) to support attachment of Salmonella enterica serovar Senftenberg, to the first and fifth to sixth true leaves and the associations between cultivar-dependent variation in plant leaf surface characteristics and bacterial attachment. Attachment levels were higher on older leaves than on the younger ones and these differences were associated with leaf vein and stomatal densities, leaf surface hydrophobicity and leaf surface soluble protein concentrations. Vein density and leaf surface hydrophobicity were also associated with cultivar-specific differences in Salmonella attachment, although the latter was only observed in the older leaves and was also associated with level of epicuticular wax.
  • Journal article
    Willing SE, Candela T, Shaw HA, Seager Z, Mesnage S, Fagan RP, Fairweather NFet al., 2015,

    Clostridium difficile surface proteins are anchored to the cell wall using CWB2 motifs that recognise the anionic polymer PSII

    , MOLECULAR MICROBIOLOGY, Vol: 96, Pages: 596-608, ISSN: 0950-382X
  • Journal article
    Aragon IM, Pérez-Mendoza D, Moscoso JA, Faure E, Guery B, Gallegos M-T, Filloux A, Ramos Cet al., 2015,

    Diguanylate cyclase DgcP is involved in plant and human Pseudomonas spp. infections

    , Environmental Microbiology, Vol: 17, Pages: 4332-4351, ISSN: 1462-2920

    The second messenger cyclic di-GMP (c-di-GMP) controls the transition between different lifestyles in bacterial pathogens. Here, we report the identification of DgcP (diguanylate cyclase conserved in Pseudomonads), whose activity in the olive tree pathogen Pseudomonas savastanoi pv. savastanoi is dependent on the integrity of its GGDEF domain. Furthermore, deletion of the dgcP gene revealed that DgcP negatively regulates motility and positively controls biofilm formation in both the olive tree pathogen P. savastanoi pv. savastanoi and the human opportunistic pathogen Pseudomonas aeruginosa. Overexpression of the dgcP gene in P. aeruginosa PAK led to increased exopolysaccharide production and upregulation of the type VI secretion system; in turn, it repressed the type III secretion system, which is a hallmark of chronic infections and persistence for P. aeruginosa. Deletion of the dgcP gene in P. savastanoi pv. savastanoi NCPPB 3335 and P. aeruginosa PAK reduced their virulence in olive plants and in a mouse acute lung injury model respectively. Our results show that diguanylate cyclase DgcP is a conserved Pseudomonas protein with a role in virulence, and confirm the existence of common c-di-GMP signalling pathways that are capable of regulating plant and human Pseudomonas spp. infections.

This data is extracted from the Web of Science and reproduced under a licence from Thomson Reuters. You may not copy or re-distribute this data in whole or in part without the written consent of the Science business of Thomson Reuters.

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