1,721,001 research outputs found
Reinvestigation into the mechanisms of Campylobacter jejuni invasion of intestinal epithelial cells
Get PDFBackground: 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.
Get PDFCampylobacter 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
Get PDFCampylobacter 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
Get PDFCampylobacter 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
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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