Identification of Avian pathogenic E. coli (APEC) genes important for the colonization of the chicken lung and characterization of the novel ExPEC adhesin I

The extraintestinal pathogen, avian pathogenic E. coli (APEC), known to cause systemic infections in chickens, is responsible for large economic losses in the poultry industry worldwide. In order to identify genes, involved in the early essential stages of pathogenesis, namely adhesion and colonization, a lung colonization model of infection was established in 5-week old White leghorn specific-pathogen free (SPF) chickens, and Signature-tagged mutagenesis (STM) was applied to this model by generating and screening a total of 1,800 mutants of an APEC strain IMT5155 (O2:K1:H5; Sequence type complex 95). The study led to the identification of new genes of interest, including adhesins, genes involved in capsule and LPS formation, regulators, transporters, metabolic enzymes and genes of putative function. Among the many genes identified was one coding for a novel APEC fimbrial adhesin (Yqi) not described for its role in APEC pathogenesis to date. Its gene product was temporarily designated ExPEC Adhesin I (EA/I). Deletion of the ExPEC adhesin I gene resulted in reduced colonization ability by APEC strain IMT5155 both in vitro and in vivo. Furthermore, complementation of the adhesin gene restored its ability to colonize epithelial cells in vitro. The ExPEC adhesin I protein was expressed as a fusion protein in E. coli BL21 in vitro during the log phase as shown by SDS-PAGE and western blotting, to reveal a protein with a molecular mass of ~ 39 kDa. Electron microscopy of an afimbriate strain E. coli AAEC189 over-expressed with the putative EA/I gene cluster revealed short fimbrial like appendages protruding out of the bacterial outer membrane. We observed that this adhesin coding gene yqi is prevalent among extraintestinal pathogenic E. coli (ExPEC) isolates, including APEC (54.4%), uropathogenic E. coli (UPEC) (65.9%) and newborn meningitic E. coli (NMEC) (60.0%), and absent in all of the intestinal pathogenic E. coli strains tested, thereby validating the designation of the adhesin as ExPEC Adhesin I. In addition, prevalence of EA/I was most frequently associated with the E. coli phylogenetic group B2 and ST95 complex of the multi locus sequence typing (MLST) scheme, with evidence of a positive selection within this highly pathogenic complex. This is the first report of the newly identified and functionally characterized ExPEC adhesin I and its role during APEC infection in chickens.