90 research outputs found
Further strategies for signature-tagged mutagenesis and the application of oligonucleotide microarrays for the quantification of DNA-tagged strains
No full textThe original signature-tagged mutagenesis (STM) technique has certain technical limitations, including high variation in signal intensities after hybridization, owing to the variability of the tag sequences, necessitating further rounds of screening. This chapter describes the basic STM approach to identify potential virulence determinants in Yersinia pseudotuberculosis and to monitor the survival rate of Helicobacter pylori mutants under different environmental conditions. In principle, this technology could be more widely applied to measure the relative abundance of tagged microbial strains in any complex environment. This chapter reasoned that the detection of DNA tagged strains could be improved by using (1) the DNA sequence tags designed to have similar hybridization properties, (2) two tags for each mutant, and (3) sequences corresponding to both strands of the tags arrayed on an affymetrix “bar coding” microarray, containing immobilized oligonucleotide tag sequences
Reinvestigation into the mechanisms of Campylobacter jejuni invasion of intestinal epithelial cells
No full textBackground: Campylobacter jejuni is an important foodborne pathogen and the leading cause of bacterial gastroenteritis. Despite the importance of C. jejuni infection and decades of research, the mechanisms for colonisation of the human intestinal tract by C. jejuni and how this causes diarrhoeal disease remain unclear, with a significant number of conflicting reports in the literature creating controversy in this area. Methods: The effect of different inhibitors of host cell pathways on the ability of the C. jejuni 81-176 wild-type strain to interact with and invade intestinal epithelial cells (IECs) was investigated. Defined isogenic C. jejuni 81-176 & 11168H ciaB, cadF and flpA mutants were constructed and characterised for the ability to interact with and invade host cells. Results: Disruption of microfilaments with Cytochalasin D increased C. jejuni invasion. Disruption of caveolae-mediated endocytosis with Methyl-beta-cyclodextrin, disruption of microtubules with Colchicine, disruption of clathrin-mediated endocytosis with Monodansylcadaverine, inhibition of phosphatidylinositol 3-kinase with Wortmannin all decreased C. jejuni invasion. Infection of the Galleria mellonella insect model with ciaB, cadF and flpA mutants resulted in a significantly reduced cytotoxic effect on the larvae. The ability of ciaB, cadF and flpA mutants to interact with and invade Caco-2 and T84 IECs was significantly reduced. The ciaB, cadF and flpA mutants exhibited a more significant decrease in the number of invasive bacteria when co-cultured with IECs in the Vertical Diffusion Chamber model. Pre-treatment of Caco-2 IECs with OMVs isolated from the ciaB, cadF and flpA mutants reduced interactions and invasion of these IECs by live C. jejuni. Conclusion: C. jejuni invasion of IECs involves modulation of many host cell pathways. CiaB, CadF and FlpA all play an important role in C. jejuni interactions with IECs leading to bacterial invasion. Further studies are still required to elucidate the exact roles that these important C. jejuni virulence factors play during interactions with and invasion of host cells
The role of the MarR transcriptional regulators RrpA and RrpB in the response of Campylobacter jejuni to oxidative and aerobic stress.
No full textCampylobacter jejuni is a microaerobic bacterium that possesses complex mechanisms to counter oxidative stress to be able to survive in the presence of reactive oxygen species (ROS). Re-annotation of the C. jejuni NCTC 11168 genome sequence identified two putative MarR-type transcriptional regulators Cj1546 and Cj1556, originally annotated as hypothetical proteins, now designated as RrpA and RrpB (regulator of response to peroxide). Both rrpA and rrpB mutants exhibit increased sensitivity to hydrogen peroxide stress compared to the wild-type strain and both mutants exhibit reduced levels of catalase (KatA) activity. However, neither mutant exhibited any significant difference in sensitivity to either cumene hydroperoxide or menadione oxidative stresses, indicating that RrpA and RrpB do not regulate expression of either alkylhydroperoxide (AhpC) or superoxide dismutase (SodB). rrpA and rrpB mutants exhibit increased biofilm formation, probably due to accumulation of ROS within the cells. Preliminary RNA-seq analysis indicated reduced katA expression in the rrpA mutant, but no differences in katA expression was observed in the rrpB mutant or rrpAB double mutant compared to the wild-type strain. C. jejuni strains normally contain rrpA, whilst only a subset contained rrpB. C. jejuni strains containing both genes were more associated with livestock-associated MLST clonal complexes. The presence of rrpB is linked to a hypervariable region containing the IF subtype of the type I Restriction-Modification (hsd) system, whereas strains containing only rrpA contain the IAB subtype hsd system. Analysis of 43 Brazilian strains identified that most chicken meat isolates contained both genes, whilst most human isolates contained only rrpA. The predominant presence of rrpB in livestock-associated C. jejuni MLST-types suggests an important role for fine-tuning oxidative stress responses through the concerted actions of multiple regulatory proteins in this microaerophilic pathogen. It also highlights the potential of genetic variation in the natural population in the adaptation to different environmental niches
Re-annotation of the Campylobacter jejuni NCTC11168 genome and functional characterisation of selected genes involved in strain pathogenesis
No full textCampylobacter jejuni is the leading bacterial cause of foodborne human gastroenteritis worldwide. The first C. jejuni genome (strain NCTC11168) was sequenced in 2000. This original annotation was a milestone in Campylobacter research, but soon became outdated. A re-annotation and re-analysis of this genome sequence was performed resulting in updates to over 90% of coding sequences (CDSs) and modification of 18.2% of CDS product functions (Gundogdu et al., 2007). Following this re-annotation, 15 uncharacterised CDSs with revised functions relating to virulence, signal transduction or regulation of gene expression were selected for further investigation. Defined isogenic C. jejuni 11168H mutants were constructed and after preliminary analysis, the Cj1556 and Cj0248 mutants were selected for further characterisation. Cj1556 was originally annotated as a hypothetical protein and was updated to a MarR family transcriptional regulator. Further bioinformatic analysis indicated a putative role in regulating the oxidative stress response. A C. jejuni 11168H Cj1556 mutant exhibited increased sensitivity to oxidative and aerobic (O2) stress, decreased ability for intracellular survival in both Caco-2 intestinal epithelial cells (IECs) and J774A.1 mouse macrophages and a reduction in virulence in the Galleria mellonella infection model. Microarray analysis of gene expression changes in the Cj1556 mutant compared to the wild-type strain indicated negative autoregulation of Cj1556 expression and down-regulation of genes associated with oxidative and aerobic (O2) stress responses. Cj0248 was originally annotated as a hypothetical protein however the re-annotation identified a HD domain linked to a superfamily of metal-dependent phosphohydrolases with roles in signal transduction in bacteria. Previously a C. jejuni 81-176 Cj0248 mutant was shown to be deficient for motility and chick colonisation, however the exact function of Cj0248 was not investigated. The C. jejuni 11168H Cj0248 mutant also possessed a reduced motility phenotype and exhibited reduced interaction and invasion when co-cultured with Caco-2 IECs compared to the wild-type strain. However the Cj0248 mutant showed no difference in autoagglutination compared to the wild-type strain and TEM analysis indicated the mutant possessed intact flagella. Higher magnification TEM indicated the possibility of an altered flagella basal body region in the Cj0248 mutant. Secretion profile analysis identified no differences in the protein profile of the Cj0248 mutant compared to the wild-type strain. The exact function of Cj0248 remains unclear
Further investigation of the roles of fibronectin-binding proteins CadF and FlpA during Campylobacter jejuni interactions with intestinal epithelial cells
No full textCampylobacter jejuni is a leading cause of bacterial gastroenteritis in humans. The ability of C. jejuni to invade human intestinal epithelial cells (IECs) is pivotal for pathogenicity. Two highly conserved fibronectin-binding proteins CadF & FlpA have been shown to play an important role in C. jejuni adhesion to IECs. CadF & FlpA are also associated with outer membrane vesicles (OMVs) and may play a role in the binding of OMVs to IECs. Mutation of cadF & flpA in 11168H & 81-176 reduced binding to fibronectin in vitro and bacterial adhesion to and invasion of both Caco-2 & T84 IECs, however intracellular bacterial numbers increased over time between 3 and 24 hours. Both cadF & flpA mutants were able to translocate as efficiently as the wild-type strain across a T84 IEC monolayer, an event not associated with any change in membrane permeability as indicated by TEER values. Mutation of cadF reduced C. jejuni cytotoxicity in the Galleria mellonella larvae model of infection to a greater extent than mutation of flpA. OMVs isolated from cadF or flpA mutants were less immunogenic and cytotoxic than OMVs isolated from wild-type strains. Results from inhibitors studies showed that cytochalasin D, methyl-beta-cyclodextrin, colchicine and wortmannin all reduced 11168H invasion of T84 IECs. However the invasion mediated by CadF and FlpA was shown to initiate different invasion pathways as wortmannin showed no effect on the ability of the 11168H cadF mutant to invade T84 IECs whilst colchicine showed no effect on the ability of the 11168H flpA mutant to invade T84 IECs. Using a 11168H strain expressing GFP at high levels, C. jejuni was shown to invade IECs, either residing within the Campylobacter containing vacuole (CCV), free within the cytoplasm and also in close proximity to parts of the trans-golgi network. A LAMP-1 stain showed co-localisation with late endosomal compartments in parts of the trans-golgi network. C. jejuni infection of IECs leads to actin cytoskeleton rearrangement as seen by the formation of filopodia, lamellapodia, membrane ruffles and F-actin stress fibres after 24 hours infection as a result of activation of the small GTPase Rac1. Mutation of cadF or flpA reduces Rac1 activation compared to wild-type strain. The significant finding of this study is that CadF and FlpA appear to initiate different invasion pathways to allow C. jejuni invasion of IECs. The results of this study support the view that both CadF and FlpA play equally important roles in C. jejuni pathogenesis
<i>C</i>. <i>jejuni</i> strain panel.
No full text<p>Strains kindly provided by</p><p>*Dr Lisa Williams, University of Bristol</p><p><sup>†</sup>Food Standards Agency, UK</p><p><sup>‡</sup>Dr Camilla Brena, University of Liverpool</p><p><sup>§</sup>Dr Nick Dorrell, London School of Hygiene and Tropical Medicine.</p><p><i>C</i>. <i>jejuni</i> strain panel.</p
Instantaneous representation and the pig itself
No full textInstantaneous representation offers the promise of showing reality itself, but as all representation removes context, it still relies on what are traditionally considered to be elements of writing, rather than existing in opposition to writing. Narrative is essential in any form of representation, as the proliferation of talk radio, 'reality television', docu-dramas, etc. shows. Virtual reality can also be seen as a type, or a continuation, of the writing process. Furthermore, editing, whether in television or photography, makes the delivery of 'reality' an idiosyncratic process rather than an impartial reporting, even in the case of instantaneous representation (i.e. live television).PT: J; CR: AUSTIN JL, 1962, SENSE SENSIBILIA BENJAMIN W, 1936, WORK ART AGE MECH RE BORGMANN A, 1999, HOLDING REALITY NYIRI JC, 1992, TRADITION INDIVIDUAL POSTER M, 1999, CYBERSPACE TEXTUALIT, P42 RING M, 1972, PERSONALIST, V53, P357 RYAN ML, 1999, SUBSTANCE, V28, P110 SELTZER M, 1998, SERIAL KILLERS SEY J, 1999, CYBERPSYCHOLOGY, P25 TIROHL B, 2000, NEW MEDIA SOC, V2, P335 VARELA EJ, 1997, EMBODIED MIND VICK RG, 1972, PERSONALIST, V53, P348; NR: 12; TC: 0; J9: NEW MEDIA SOC; PG: 10; GA: 631TNSource type: Electronic(1
Campylobacter jejuni modulates reactive oxygen species production and NADPH oxidase 1 expression in human intestinal epithelial cells
No full textCampylobacter jejuni is the major bacterial cause of foodborne gastroenteritis worldwide. Mechanistically, how this pathogen interacts with intrinsic defence machinery of human intestinal epithelial cells (IECs) remains elusive. To address this, we investigated how C. jejuni counteracts the intracellular and extracellular reactive oxygen species (ROS) in IECs. Our work shows that C. jejuni differentially regulates intracellular and extracellular ROS production in human T84 and Caco-2 cells. C. jejuni downregulates the transcription and translation of Nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase (Nox1), a key ROS-generating enzyme in IECs and antioxidant defence genes cat and sod1. Furthermore, inhibition of Nox1 by diphenylene iodonium (DPI) and siRNA reduced C. jejuni ability to interact, invade and intracellularly survive within T84 and Caco-2 cells. Collectively, these findings provide mechanistic insight into how C. jejuni modulates the IEC defence machinery
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Retrospective thermal neutron fluence determination using lithium-ion mobile telephone batteries
No full textFortuitous dosimeters are radiosensitive objects carried by an individual who was exposed to radiation. These objects can be analyzed some time after exposure and the results can be used to aid in calculating radiation fields and doses received by individuals. Items that make good fortuitous dosimeters are those that are consistent in their manufacture and are carried by a large percentage of the population. Some materials are more suitable than others for retrospective dosimetry, depending upon their sensitivity, signal retention (i.e. fading), and the type of radiation to which they respond. The effectiveness and sensitivity of lithium-ion (Li-ion) mobile telephone batteries as fortuitous neutron dosimeters is investigated in this research. Neutron fluences are estimated based upon the activation products formed in batteries during exposure. In the past, objects such as keys and coins were used for retrospective neutron dosimetry.
Mobile phone batteries were chosen as possible candidates for dosimeters because of their widespread use by the general population and the observation that most users carry these objects on or near the torso.
Lithium-ion mobile-phone batteries were irradiated with neutrons in beam port #4 of the Oregon State University TRIGA® reactor and subsequently analyzed using gamma spectroscopy in order to identify activation products formed, and to determine the linearity of their response to several neutron fluences. Batteries exposed to thermal neutron fluencies ranging from 2.8x10⁸ n·cm⁻² to 3.4x10¹⁰ n·cm⁻² generated ⁶⁰Co count rates ranging from 0.022 (±4.5%) cps to 2.890 (±0.6%) cps. Cobalt emerged as an element commonly found in Li-ion battery cathodes as a candidate for neutron activation analysis months to years following their irradiation. This is due to the relatively high cobalt content by weight found in these batteries and its 5.27 year half-life. Thermal neutron fluences were estimated with accuracy ranging from 2% to 23% of actual fluences, with an average accuracy of 12%. The activation of cobalt in the samples was highly linear with fluence (R²=0.995)
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