Novel roles for autotransporter adhesin AatA of avian pathogenicEscherichia coli: colonization during infection and cell aggregation

Abstract

Systemic infections in avian species caused by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide. To unravel factors possibly involved in APEC pathogenicity, suppression subtractive hybridization was applied, leading to the identification of a putative APEC autotransporter adhesin gene aatA in our previous study. In this study, pathogenic mechanism of AatA was further determined. A deletion mutant of aatA was constructed in the APEC DE205B, which results in the reduced capacity to adhere to DF-1 cells, defective virulence in vivo, and decreased colonization capacity in lung during the systemic infection compared with the wild-type strain. Furthermore, these capacities were restored in the complementation strains. These results indicated that AatA makes a significant contribution to APEC virulence through bacterial adherence to host tissues in vivo and in vitro. In addition, aggregation assays for strain AAEC189 expressing aatA indicated that AatA mediates cell aggregation and settling of cells. However, this cell aggregation is blocked by Type I fimbriae. This study illustrates the first examination of the role of AatA in aggregation and systemic infection.

Characterization and Functional Analysis of AatB, a Novel Autotransporter Adhesin and Virulence Factor of Avian Pathogenic Escherichia coli

“…The APEC strain DE205B was isolated from the brain of a duck with septicemia and neurological symptoms and belonged to the phylogenetic E. coli reference (ECOR) group B2, as determined by phylogenetic analysis using multiplex PCR (37,38). This strain harbored the virulence-associated genes tsh, mat, fyuA, irp2, iucD, iutA, iss, aatA, aatB, vat, malX, fimC, ompA, ibeA, ibeB, yijp, gimB, and aslA but was negative for papC and hlyA by PCR analysis (38)(39)(40). Strain DE205B was used for infection studies, mutant construction, and functional assays.…”

“…The adhesion capacities of wild-type strain DE205B, mutant strain DE205B⌬aatB, and complementation strain DE205BC⌬aatB were studied with cultured chicken embryo fibroblasts, as previously described (23,39). Briefly, semiconfluent monolayers of DF-1 cells were washed and incubated with Dulbecco's modified Eagle's medium (DMEM) without fetal bovine serum containing bacteria at a multiplicity of infection of 100.…”

“…For immunoblot analysis, protein samples were loaded into 12% SDS gels and transferred onto a polyvinylidene fluoride membrane (Amersham Pharmacia Biotech, Uppsala, Sweden) using a semidry blotting apparatus (TE77; Amersham Pharmacia Biotech), as described previously (23,38,39). Anti-AatB and anti-DE205B immune sera were used as primary antibodies.…”

“…The resulting plasmid, pET28a-aatB, was transformed into competent E. coli BL21(DE3) cells, and the AatB protein was expressed by isopropyl-beta-D-thiogalactopyranoside (IPTG) induction at a final concentration of 1 mM. Protein purification was performed Polyclonal anti-AatB antiserum was raised in New Zealand White rabbits by subcutaneous immunization, as described previously (23,38,39). The immunization dose was 300 g of purified AatB protein mixed with an equal volume of Montanide ISA 206 adjuvant (SEPPIC, Lyon, France).…”

“…For complementation studies, the aatB operon, including its putative promoters, was amplified and subcloned into the pUC18 vector using the primer pair ZGXK5F and ZGXK6R with restriction enzyme recognition sites EcoRI and BamHI, as described previously (23,38,39). The colonies were tested for the presence of aatB.…”