|163-1||In vitro Selection of Non-invasive Avian Pathogenic Escherichia coli Mutants Generated by Signature-tagged Mutagenesis|
|Autores:||Daniel Brisotto Pavanelo (UFRGS - Universidade Federal do Rio Grande do Sul) ; Leticia Beatriz Matter (UFSM - Universidade Federal de Santa Maria) ; Fabiana Horn (UFRGS - Universidade Federal do Rio Grande do Sul) |
Avian Pathogenic Escherichia coli is the causative agent of colibacillosis in poultry, a disease that can be localized or systemic and have a variety of symptoms. The most severe manifestation of the infection is colisepticemia, believed to be initiated by colonization of the avian lung. However, how bacteria reaches the bloodstream is unclear. In a previous work, we tested adhesion and invasion of eight APEC strains to avian fibroblasts, and among those strains only MT78 was capable of invading these cells. MT78 harbor the same virulence-associated genes of other non-invasive APEC strains, raising the possibility that there are other genes – not yet described – involved in invasion of these non-phagocytic cells.
To understand the mechanism by which MT78 invades non-phagocytic cells, we propose the use of signature-tagged mutagenesis (STM) to generate non-invasive mutants, and to identify the correspondent disrupted genes. Using this technique, we generated 1,800 mutants of MT78 by randomly inserting tagged-transposons in the bacterial genome. For that, 90 pUTmini-Tn5km2 plasmids with different tagged-transposons were inserted into strain S17-1 λ pir through electroporation. S17-1 λ pir with different plasmids were conjugated to MT78, and 20 colonies were selected in each conjugation, totalizing 1,800 mutants (90 tags x 20 colonies).
Each pool, containing 90 MT78 mutants (input pool), was then tested in the invasion assay to CEC-32 avian fibroblasts to select for non-invasive phenotype. The recovered, invasive mutants represent the output pool. The non-invasive mutants – absent from the output pool – will be identified by comparing the DNA extracted from the input pool with that from the output pool. This comparison will be done through dot blot, using the mutant tags as a bar code for identification. We will then identify the disrupted gene in the non-invasive mutants by sequencing the genome region randomly targeted by the tagged-transposon.
Palavras-chave: Avian pathogenic E. coli (APEC), Extraintestinal pathogenic E. coli ExPEC, Signature-tagged mutagenesis (STM), Avian fibroblasts CEC-32, Invasion to eucaryotic cells