The use of RIVET to identify avian pathogenic Escherichia coli (APEC) genes induced in vivo during infection in chickens

Abstract

Event Abstract Back to Event The use of RIVET to identify avian pathogenic Escherichia coli (APEC) genes induced in vivo during infection in chickens Huruma N. Tuntufye1* 1 Catholic University of Leuven, Faculty of Bioscience Engineering, Belgium H. N. TUNTUFYE1, 2, S. LEBEER3 and B. M. GODDEERIS1 1Department of Biosystems, Faculty of Bioscience Engineering, Katholieke Universiteit Leuven, Kasteelpark Arenberg 30, B-3001, Heverlee, Belgium 2Department of Veterinary Microbiology and Parasitology, Sokoine University of Agriculture, P.O.Box 3019, ChuoKikuu, Morogoro, Tanzania 3Department of Microbial and Molecular Systems, Faculty of Bioscience Engineering, Katholieke Universiteit, Kasteelpark Arenberg23, 3001, Heverlee, Belgium Avian pathogenic Escherichia coli (APEC) are a group of E.coli strains causing systemic disease in poultry known as avian colibacillosis. The disease manifests itself initially with septicaemia then either sudden death or localized multiple organ inflammation. The disease is associated with major economic losses to the poultry industry worldwide. Host and bacterial factors influencing and/or responsible for carriage and systemic translocation of APEC inside the host are poorly understood. Identification of such factors could help in the understanding of its pathogenesis and subsequently development of control strategies. In this study RIVET strategy (Camilli et al., 1994) was developed and used to isolate host-induced APEC promoters in order to investigate APEC pathogenesis in chicken. Random chromosomal DNA fragments from APEC genome were transcriptionally fused upstream to a promoterless cre gene to create an APEC RIVET library in a promoter trap plasmid. The reporter strain was constructed by integrating the loxP sites (in direct orientation) flanking the neomycin resistance marker (neo) for positive selection and streptomycin sensitivity gene (rpsL) for negative selection into APEC genome. Fused active promoters cause expression of Cre recombinase which subsequently cause recombination of the two loxP sites, deleting the cassette and permanently changing the bacterial phenotype such that could be detected after gene expression had ceased. The APEC RIVET library was pre-selected on kanamycin and ampicillin antibiotics to eliminate in vitro active promoters. The bacteria were then administered in chicken host via intra-tracheal route. After screening, 147 kanamycin sensitive clones were isolated from chicken indicating that the loxP-rpsL-neo-loxP (LoxP) cassette could be deleted as a result of in vivo active promoters. PCR analysis and sequencing demonstrated a range of insert sizes (1-2kb) suggesting that the screening is functional and the plasmid could stably be maintained in the bacteria even after infection. Further analysis of the DNA fragments' sequences revealed that 27 different APEC genes were induced inside the chicken during infection. The in vivo induced genes include metabolic genes, virulence genes and genes with prophage functions, regulatory functions and unknown functions. With these results APEC RIVET library could be adapted and the strategy showed to be functional for the screening of host-induced APEC genes/ promoters in chickens. Keywords: APEC, Chickens, RIVET strategy Conference: ECMIS - E. coli and the Mucosal Immune System : Interaction, Modulation and Vaccination, Ghent, Belgium, 2 Jul - 5 Jul, 2011. Presentation Type: Oral Presentation Topic: Infection and innate immunity, immunosupression and/or immunostimulation Citation: Tuntufye HN (2012). The use of RIVET to identify avian pathogenic Escherichia coli (APEC) genes induced in vivo during infection in chickens. Front. Immunol. Conference Abstract: ECMIS - E. coli and the Mucosal Immune System : Interaction, Modulation and Vaccination. doi: 10.3389/conf.fimmu.2012.01.00009 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 09 Jan 2012; Published Online: 12 Jan 2012. * Correspondence: Mr. Huruma N Tuntufye, Catholic University of Leuven, Faculty of Bioscience Engineering, Heverlee, 3001, Belgium, Huruma.Tuntufye@biw.kuleuven.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Huruma N Tuntufye Google Huruma N Tuntufye Google Scholar Huruma N Tuntufye PubMed Huruma N Tuntufye Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.