Biomedical subjects

Ian Connerton

Publications and source records attributed to Ian Connerton.

7 recordsLinked to original sources

PubMed2007

Improved growth of enteric adenovirus type 40 in a modified cell line that can no longer respond to interferon stimulation.

Human enteric adenoviruses propagate poorly in conventional human cell lines used to grow other adenovirus serotypes. As human enteric adenoviruses have a defect in counteracting the cellular interferon (IFN) response in cell culture, to aid in growth of the virus, a 293-based cell line defective in its ability to respond to IFN was constructed. This cell line (293-SV5/V) constitutively expresses V-protein of the paramyxovirus Simian virus 5, which degrades the signal transducer and activator of transcription 1 (STAT1) and thereby prevents the STAT1-mediated IFN response. Analysis of human enteric adenovirus type 40 (HAdV-40)-infected 293-SV5/V cells compared with parental 293 cells shows that the recombinant line allows more rapid production of virus and results in higher titres. These results suggest that the defect in HAdV-40 in counteracting the IFN response can be overcome at least partially through the use of 293-SV5/V cell lines.

Adenoviruses, Human↗

PubMed2006

The enteropathogenic Escherichia coli type III secretion system effector Map binds EBP50/NHERF1: implication for cell signalling and diarrhoea.

Enteropathogenic Escherichia coli (EPEC) is the single most important contributor to child diarrhoea in developing countries. Nevertheless, the mechanism responsible for EPEC diarrhoea remains elusive. Using the yeast two-hybrid system to determine the target host cell protein of the EPEC type III secretion system effector Map led to identification of ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50), also known as Na+/H+ exchanger regulatory factor 1 (NHERF1). Protein interaction is mediated by the carboxy-terminal Thr-Arg-Leu (TRL) motif of Map and the PSD-95/Disk-large/ZO-1 domain 1 (PDZ1) of EBP50/NHERF1. Although EBP50/NHERF1 is recruited to site of EPEC adhesion in a Map-independent mechanism, co-immunoprecipitation and immunostaining revealed that Map binds to, induces proteolysis of, and colocalizes with EBP50/NHERF1 during infection of cultured epithelial cells. The TRL motif of Map was involved in Map-induced filopodia formation and brush border elongation on infected HeLa and Caco-2 cells respectively. As EBP50/NHERF1 regulates ion channels in the intestine we assessed the involvement of Map in diarrhoea using the Citrobacter rodentium mouse model of EPEC. We report significantly greater diarrhoea following infections with wild-type C. rodentium compared with C. rodentiumDeltamap. These results provide new insights into the mechanisms of EPEC diarrhoea.

Actins↗

PubMed2006

Host protein interactions with enteropathogenic Escherichia coli (EPEC): 14-3-3tau binds Tir and has a role in EPEC-induced actin polymerization.

Enteropathogenic Escherichia coli (EPEC) cause infantile diarrhoea and are characterized by their ability to produce attaching and effacing lesions on the surface of intestinal epithelial cells. EPEC employ a filamentous type III secretion system to deliver effector molecules that subvert mammalian cell function to generate actin- and cytokeratin-rich pedestals beneath adherent bacteria. Tir is a major effector protein that is delivered to the plasma membrane of the eukaryotic cell where it acts as the receptor for the bacterial adhesin intimin. Host cell proteins that are recruited to the site of intimate attachment include focal adhesion and cytoskeletal proteins that contribute to pedestal formation. We have used Tir as bait in a yeast two-hybrid screen to identify the protein 14-3-3tau as a binding partner. 14-3-3 proteins are a family of adaptor proteins that modulate protein function in all eukaryotic cells. Here we demonstrate that the tau isoform (also known as theta) of 14-3-3 can bind specifically to Tir in a phosphorylation-independent manner, and that the interaction occurs during the infection process by co-immunoprecipitation of the partners from infected HeLa cell extracts. 14-3-3tau is recruited to the site of the pedestal (3 h after infection) and can decorate attached EPEC in the later stages of the infection process (5-7 h). Pedestal formation can be impaired by depletion of cellular 14-3-3tau using small interfering RNAs. This study indicates a direct functional role for the 14-3-3tau:Tir interaction and is the first to demonstrate the association of a host protein with the surface of EPEC.

14-3-3 Proteins↗

PubMed2006

EspF of enteropathogenic Escherichia coli binds sorting nexin 9.

EspF of enteropathogenic Escherichia coli targets mitochondria and subverts a number of cellular functions. EspF consists of six putative Src homology 3 (SH3) domain binding motifs. In this study we identified sorting nexin 9 (SNX9) as a host cell EspF binding partner protein, which binds EspF via its amino-terminal SH3 region. Coimmunoprecipitation and confocal microscopy showed specific EspF-SNX9 interaction and non-mitochondrial protein colocalization in infected epithelial cells.

Amino Acid Motifs↗

PubMed2005

Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening.

The functional expression of olfactory receptors (ORs) is a primary requirement to examine the molecular mechanisms of odorant perception and coding. Functional expression of the rat I7 OR and its trafficking to the plasma membrane was achieved under optimized experimental conditions in the budding yeast Saccharomyces cerevisiae. The membrane expression of the receptor was shown by Western blotting and immunolocalization methods. Moreover, we took advantage of the functional similarities between signal transduction cascades of G protein-coupled receptor in mammalian cells and the pheromone response pathway in yeast to develop a novel biosensor for odorant screening using luciferase as a functional reporter. Yeasts were engineered to coexpress I7 OR and mammalian G(alpha) subunit, to compensate for the lack of endogenous Gpa1 subunit, so that stimulation of the receptor by its ligands activates a MAP kinase signaling pathway and induces luciferase synthesis. The sensitivity of the bioassay was significantly enhanced using mammalian G(olf) compared to the G(alpha15) subunit, resulting in dose-dependent responses of the system. The biosensor was probed with an array of odorants to demonstrate that the yeast-borne I7 OR retains its specificity and selectivity towards ligands. The results are confirmed by functional expression and bioluminescence response of human OR17-40 to its specific ligand, helional. Based on these findings, the bioassay using the luciferase reporter should be amenable to simple, rapid and inexpensive odorant screening of hundreds of ORs to provide insight into olfactory coding mechanisms.

Biological Assay↗

PubMed2004

Involvement of the intermediate filament protein cytokeratin-18 in actin pedestal formation during EPEC infection.

While remaining extracellular, enteropathogenic Escherichia coli (EPEC) establish direct links with the cytoskeleton of the target epithelial cell leading to the formation of actin-rich pedestals underneath attached bacteria. The translocated adaptor protein Tir forms the transmembrane bridge between the cytoskeleton and the bacterium; the extracellular domain of Tir functions as a receptor for the bacterial adhesin intimin, while the intracellular amino and carboxy termini interact with a number of focal adhesion and other cytoskeletal proteins; and recruitment of some is dependent on phosphorylation of Tyr 474. Using Tir as bait and HeLa cell cDNA library as prey in a yeast two-hybrid screen, we identified cytokeratin 18 as a novel Tir partner protein. Cytokeratin 18 is recruited to the EPEC-induced pedestal and has a direct role in actin accretion and cytoskeleton reorganization. This study is the first to implicate intermediate filaments in microfilament reorganization following EPEC infection.

Actins↗

PubMed2003

Engineered yeasts as reporter systems for odorant detection.

The functional expression of olfactory receptors (ORs) is a primary requirement to utilize olfactory detection systems. We have taken advantage of the functional similarities between signal transduction cascades in the budding yeast Saccharomyces cerevisiae and mammalian cells. The yeast pheromone response pathway has been adapted to allow ligand-dependent signaling of heterologous expressed G-protein coupled receptors (GPCRs) via mammalian or chimeric yeast/mammalian Galpha proteins. Two different strategies are reported here which offer a positive screen for functional pairs. The OR and Galpha protein are introduced into the modified yeast cells such that they hijack the pheromone response pathway usually resulting in cell cycle arrest. The first strategy utilizes ligand-induced expression of a FUS1-HIS3 reporter gene to permit growth on a selective medium lacking histidine; the second to induce ligand-dependent expression of a FUSI-Hph reporter gene, conferring resistance to hygromycin. Validation of the systems was performed using the rat 17 receptor response to a range of aldehyde odorants previously characterized as functional ligands. Of these only heptanal produced a positive growth response in the concentration range 5 x 10(-8) to 5 x 10(-6) M. Induction conditions appear to be critical for functional expression, and the solvents of odorants have a toxic effect for the highest odorant concentrations. The preference of rat 17 receptor for the ligand heptanal in yeast has to be compared to concurrent results obtained with mammalian expression systems.

Animals↗